TW200900079A - Methods of treating ophthalmic diseases - Google Patents

Abstract

Methods of using inhibitors (including monoclonal antibodies) directed against amyloid-beta peptide for the treatment of ophthalmic diseases such as age-related macular degeneration are described.

Description

200900079 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a method for using an antibody against a starch-like peptide, which is used for treating and/or preventing an ophthalmic disease such as age-related macular degeneration, and is used for Other eye diseases, such as glaucoma, diabetic retinopathy (including diabetic macular edema), choroidal neovascular membrane (CNV) uveitis, myopia degeneration, eye tumor, central retinal vein occlusion (central retinal vein 〇cclusi〇n) ), iris reddening, ocular neovascularization, central seric retinopathy (central ser_retniopathy), surface problems such as dry eye (〇cuiar d1SCUS), central retinal artery occlusion, cystic macular edema, and any other retinal degeneration Sexual disease. The present application claims the benefit of U.S. Patent Application Serial No. 12/41,581, filed on March 3, 2008, which is incorporated herein by reference. , right of 181, the entire contents of which is incorporated herein by reference. [Prior Art] In the United States, the most common corrected visual acuity in individuals over the age of 65 is caused by a retinal disorder called age-related macular degeneration (AMD). As AMD progresses, the disease is characterized by a sharp, loss of central vision. The area of the eye affected by AMD is a small area of the macula _ retinal center, which is mainly composed of photoreceptor cells. It accounts for about 85%-90°/ of AMD patients. The so-called "dry AMD (also known as "map-like atrophy (Lv (3) to change the eye pigment distribution caused by the overall atrophy of the cells, photoreceptor loss and view 129629.doc 200900079, '.*, force this reduction >, s stomach "wet type" AMD involves abnormal choroidal angiogenesis that causes coagulation or obstruction in the space under the optic net. Therefore, the onset of wet AMD forms an abnormal choroidal neovascular network under the nerve retina. (choroidal neovascularization; t && .. Wang attack CNV) occurs. The newly formed blood vessels are excessively infiltrated. This leads to subretinal fluid and blood accumulation, causing loss of visual acuity. Finally, when it involves choroid and When a large disc-shaped scar of the retina is formed, there is a total loss of functional retina in the area involved. Although dry-type sputum patients can maintain a reduced quality vision, wet AMD often causes blindness. (Hamdi A Kenney, Age-related Macular degeneration-a new W (4) is called Fr State M (10)e3〇53i4, 2〇〇3 years). CNV occurs not only in wet AMD but also in other ocular pathology, these other eye diseases Such as glaucoma, diabetic retinopathy (including diabetic macular edema), Bruch's membrane rupture, myopia degeneration, eye tumors and other related retinal degenerative diseases. The pathogenesis of AMD is obviously multi-factorial Common disorders in which genetic and environmental factors play a role in their onset and progression. Various studies have identified several risk factors for AMD, such as smoking, aging, and family history (Milton, / 88, 269 (1979); MhcheU et al,

Ophthalmology 102, 1450-1460 (1995) ; Smith et al, (9ρ/ζί/ζα/则/〇烈108, 697-704 (2001)), gender (7 times more than women in the month of women) Klein , 99, 933-943 (1992)) and race (whites are most vulnerable) ^ Other risk factors may include eye characteristics such as farsightedness, hyperopia, and light-colored eyes, as well as cardiovascular disease and hypertension. Proof of genetic involvement in the progression of disease 129629.doc 200900079 It has also been documented (see Ham di and Kenney above). There is currently no universally accepted animal model for studying AMD. The initial study by Malek et al. (pNAS 102, 119〇〇_5 (2〇〇5)) has produced animal models with three risk factors with morphological features that approximate human ΑΜΕ when combined. It is worth noting that the development of this mouse model has provided an opportunity for amd to measure the novel molecular mechanisms of D and the therapeutic goals. There is still a need to identify novel targets and therapeutic agents that can treat and/or prevent the following ophthalmic diseases: such as age-related macular degeneration (wet and dry), glaucoma, diabetic retinopathy (including diabetic macular edema), choroid Neonatal meninges (CNV) _ 臈 臈 inflammation, myopia degeneration, eye tumors, central retinal vein occlusion, iris reddening, ocular neovascularization, central serous retinopathy, surface problems such as dry eye, central retinal artery occlusion , cystic macular edema and other retinal degenerative diseases. SUMMARY OF THE INVENTION V./* The present invention discloses a novel therapeutic method associated with the pathogenesis of an ophthalmic disease. The present invention discloses a method for treating an ophthalmic disease, which comprises administering an effective amount of an inhibitor to an individual. Starch-like (Ap) peptide. Inhibitors can be administered to individuals with ophthalmic diseases such as age-related macular degeneration (wet and dry 'AMD'), glaucoma, diabetic retinopathy (including diabetic yellow stagnation), choroidal neovascularization : myopia degeneration, eye tumor, central retinal vein occlusion, rainbow angiography, neovascularization, central sputum ocular lesions, surface problems such as dry eye, retinal dysplasia Obstruction, cystic macular edema and its disease. In the examples, the inhibitor is an antibody, an anti-129629.doc 200900079 molecule, an siRNA molecule, a nuclear spinosa, a ribonuclease or a small molecule compound. In one embodiment, the present invention is a method of treating an individual having an age-degraded individual, and wherein the sentence is treated with a therapeutically effective amount of β- A coating of the type of powder (Αβ)-like inhibitor & Another embodiment of the present invention relates to a treatment * 右生私J. A method of treating an individual having age-related macular degeneration (AMD) comprising administering to the individual a pharmaceutical composition comprising, i 3 , an orally effective amount of an A beta inhibitor. Another embodiment of the present invention provides a use of a sputum/alpha therapeutically effective amount of an Αβ inhibitor for the preparation of a sputum sputum xm smear M/ for promoting rehabilitation of a patient having AMD drug. In one aspect of this embodiment, the genus I 3 has an F c region that impairs the effector function. In another sad example of this embodiment, the disease is AMD, including thirsty and dry AMD. The present invention also provides a method of treating or preventing a disease associated with (d) powder deposition, and comprising administering to a subject an effective amount of a pharmaceutical composition comprising a specific binding to an aβ peptide or an ap peptide aggregate form. antibody. In another aspect of this embodiment, the antibody comprises an Fe region with a variation in the naturally occurring & region, wherein the mutation causes a weakening effect function. In some embodiments, the administration of the antibody produces microbleeds that are smaller than the administration of the antibody without mutation. The antibodies and polypeptides useful in the methods of the invention specifically bind to a Αβ peptide or a learned aggregate form. In the examples, the antibody or polypeptide has attenuating effect stress. In the actual case, the antibody or polymorphism is not the 仏,) 2 fragment. In the second embodiment, the antibody or polypeptide is not a Fab fragment. In some embodiments, the antibody or polypeptide is not a single chain antibody scFv. 129629.doc 200900079 A polypeptide that specifically binds to an aggregate form of an Αβ peptide or a Αβ peptide and that comprises a heavy chain constant region with attenuating effector function can also be used in any of the methods described herein. In some embodiments, the polypeptide comprises a derivative. Sequences (e.g., one or more CDRs) of the variants thereof shown in antibodies 9TL, 6G or Table 3 or Table 8. In some embodiments 'antibody or polymorphism comprises a heavy chain constant region having a weakening effector function, wherein the heavy chain constant region comprises an Fc region. In some embodiments, the N-glycosylation in the Fc region is removed. In some embodiments, the Fc region comprises a mutation within the N-glycosylation recognition sequence whereby the Fc region of the antibody or polypeptide is not glycosylated. In some embodiments, the Fc region is PEGylated. In some lambda applications, the heavy chain constant region of the antibody or polypeptide is the human heavy bond IgG2a definitive region. The constant region contains the following mutation: Α33〇ρ33ι to 83383831 (Refer to wild type 1 § (32& sequence amino acid numbering). In some examples, the antibody or polypeptide comprises 1 § (the constant region of 34, which contains the following mutations: E233F234L235 to P233V234A235. In an embodiment, the antibody or polypeptide specifically binds to an epitope within the residue of the Aβ peptide. In some embodiments, the antibody or polypeptide specifically binds to the terminus of the Aβ peptide. In some embodiments The antibody or polypeptide specifically binds to an epitope within residues 16-28 of the Aβ peptide. In some embodiments, the antibody specifically binds to an epitope on the C-terminal side of the Aβ peptide, such as by an amino acid. 25 or an amino acid-initiating epitope. The antibody can be combined with any of the Αβ peptides 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43. Specific binding. In some embodiments, the antibody can be truncated with a c-terminal Αβ peptide Specific binding to the C-terminal amino acid, such as Αβ 1_37, 129629.doc 200900079, WO, 1-41, 1-42, 1-43. In one embodiment, the antibody or oxime peptide and Αβ, 'peptide The antigenic determinant specifically binds. In another aspect of this embodiment, the antibody or polypeptide specifically binds to an epitope on the _42 peptide. In the re-situation of this embodiment, the antibody or multi-^ The antigenic determinant-specific binding of Αβ. In some embodiments, the antibody or polypeptide is specific to Αβι, the residue of peptide 28, and the antigenic thiol group is specific. In the actual case, the antibody or polypeptide and the AJ3i42 peptide are The epitope in the residue 42 specifically binds. In some embodiments, the antibody or polypeptide specifically binds to an epitope within the residue 2" of the Αβ, _43 peptide. In some embodiments, the antibody Or the polypeptide specifically binds to the Αρ peptide, and the second full-length starch-like precursor protein (ΑΡΡ) binds. In some embodiments, the plastid or polypeptide specifically binds to the aggregated form of Αβ, but not to the soluble form: In an embodiment, the antibody or polypeptide is specifically associated with a soluble form of Aβ And not in combination with the aggregated form. In some embodiments, the antibody or polypeptide specifically binds to the aggregated form and soluble form of Aβ. In some embodiments, the antibody or polymorphism is associated with Ah. 4〇Specific binding. In some embodiments, the antibody or polypeptide specifically binds to an epitope comprising 胺β" of the amino acid 35-40. In some embodiments, the antibody or polypeptide comprises an amine The amino acid of 36_4G is 抗原βι, and the epitope is specifically bound. In some implementations, the antibody or polypeptide and 包括β,.* including amino acid 39 and/or 4G. The antigen on the substrate is specifically bound. In some embodiments, the antibody or polypeptide specifically binds to Ah, but does not: Αβ〗 42 and/or Αβ^3 specifically bind. In some embodiments, the antibody comprises a variable region of an antibody 9TL described herein or an antibody derived from 9TL. In 129629.doc -10- 200900079, in some cases, antibodies or genomically inhibited antibodies, 6G and/or antibodies or polypeptides derived from 9TL or 6G and individual Αβ peptides are used by gentlemen. In some embodiments, the antibody or polypeptide binds to Αβυο with a higher affinity than its binding to APm and Αβΐ-43. In another aspect of this embodiment, the antibody is not antibody 2294. In some embodiments, the antibody binds to an epitope comprising the amino acid 25-34 and 40. In some embodiments, the antibody comprises a variable region of antibody 6G or an antibody derived from 6〇} as described herein. In some embodiments, the antibody or polypeptide competitively inhibits binding of antibody 6 (or) and/or antibody or polypeptide derived from 6G to Aβ. In some embodiments, the antibody or polypeptide is about 1〇〇η or less, or 2〇ηΜ or below, or a binding affinity of 2 ηΜ or less (reverse 〇) binding to the octapeptide. In the aspect of this embodiment, the antibody or polypeptide is about 1 〇〇 or less, 50 ηΜ or Hereinafter, or 2 η Μ or below, "in combination with hydrazine. In another aspect of this embodiment, the antibody or polymorphism is also about _ Å or less ' 50 η Μ or less, or 2 福 or less κ 〇 Ah. Peptide Binding. Antibodies or polypeptides that bind to the Αρ peptide can be administered by any of the methods known in the art, including: intravenous, subcutaneous, via inhalation, intra-arterial, intracardiac, intraventricular, non-menstrual Intestinal, intrathecal, and intraperitoneal. It can be administered by a second dose; the main shot and/or can be systemic (for example, intravenously) or topically administered. This is also generally applicable to the polypeptide of the present invention and polynucleotide. Ming also provides the means to treat ophthalmic diseases by administering pharmaceutical compositions: The composition comprises an effective amount of an antibody or a polypeptide which binds to the aggregation of the Αρ peptide or the partial peptide and which has a weakening effect function, or a polynucleotide which encodes the antibody or polypeptide, and the pharmaceutically acceptable 129629 .doc 200900079 Accepted Excipients. The invention also provides kits and compositions comprising any one or more of the compositions comprising an effective amount of specific binding to an aggregated form of aprotinin or aβ peptide Any of the antibodies or polypeptides, or polynucleotides encoding the antibodies or polypeptides. One of the kits in a suitable package and having appropriate instructions applies to the methods described herein. The present invention also provides a method for producing a m-antibody for treating a disease associated with deposition of a powder such as a human peptide, the method comprising selecting and Ap a first humanized antibody to which the peptide specifically binds; and a modified humanized antibody that alters the antibody to provide a weakening effect relative to the first humanized antibody. Another embodiment of the present invention (4) is directed to protection or Restore A method of individual retinal function comprising administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of a wall formulation. In one embodiment, the inhibitor is an antibody, an antisense molecule, an siRNA molecule, a ribonuclease or a small molecule compound The present invention is directed to a method of maintaining or restoring an individual's visual acuity 'which comprises a therapeutically effective amount of an A beta inhibitor. In one aspect of the above embodiments, the above method is used for Inappropriate also in individuals treated with Alzheimer's disease, D〇wn, s syndrome or brain amyloplasty. The above method of the invention includes an Αβ inhibitor as an antibody In one aspect, the invention disclosed herein relates to an antibody that binds to the C-terminus of Ah, a peptide (SEQ ID ΝΟ: 15 shown in Table 4). Thus, in one aspect, the method comprises treatment with antibody 9TL (interchangeably referred to as "9TL,") which has ATCC accession numbers PTA-6124 and PTA-6125 by means of ATCC accession numbers PTA-6124 and PTA-6125 The expression vector is produced. The amino acid sequence of the heavy and light chain variable regions of 9TL is shown in Figure 1. The complementary decision region (CDR) portion of antibody 9TL (including Chothia and Kabat CDRs) is also shown in Figure 1. It is understood that references to any portion or whole of the 9TL region encompass sequences generated by a performance vector having ATCC accession numbers PTA_6124 & pTA_6l25, and/or sequences depicted in the figures. In another aspect, the invention encompasses An antibody variant having a 9TL having the amino acid sequence depicted in Table 3 is administered. In another aspect, the invention comprises administering a fragment or region comprising the antibody 9TL or the variant thereof shown in Table 3. In one embodiment, the fragment is the light chain of antibody 9TL. In another embodiment, the fragment is the heavy chain of antibody 91. In yet another embodiment, the fragment contains the light chain from antibody 9 / or one or more variable regions of the heavy chain. In yet another embodiment, The fragment contains one or more variable regions from the light and/or heavy chains shown in Figure 1. In yet another embodiment, the fragment contains one or more of the light and/or heavy chains from the antibody 9TL In another aspect, the invention comprises administering a polypeptide (which may or may not be an antibody) comprising any one or more of the following: a) an antibody or a variation thereof as shown in Table 3. One or more (: £>11; b) cdr H3 from the heavy chain of the variant shown in antibody 9tl or Table 3; c) from the antibody 9TL or its variant shown in Table 3 CDR L3 of the light chain; d) three CDRs from the light chain of the variant 9TL or the variant shown in Table 3; b) three heavy chains from the antibody 9TL or the variants thereof shown in Table 3 CDR; f) three CDRs from the light chain of the variant 9TL or the variant shown in Table 3 and the heavy chain from the 129629.doc-13-200900079 antibody 9TL or the variants thereof shown in Table 3 Three CDRs. The invention further provides for administering a polypeptide (which may or may not be an antibody) comprising any one or more of the following: a) derived from antibody 9TL or as shown in Table 3. One or more (one, two, three, four, five or six) CDRs of the variant; b) a CDR derived from the heavy chain of the antibody 9TL; and/or c) derived from the antibody 9TL The CDRs of the CDRs of the light chain. In some embodiments, the CDRs are the cDRs shown in Figure 1. In some embodiments, the or the CDRs derived from the variants of the antibody 9TL or Table 3 are shown. At least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 9%, at least about 91%, at least about 92%, at least about 93%, at least about 94% At least about 95°/. At least about 96°/. At least about 970/. At least about 98% or at least about 99% is at least one, at least two, at least three, at least four, at least five, or at least six C D R' of 9TL or a variant thereof. In another aspect, the invention comprises administering antibody 6G (interchangeably referred to as "6G"). Figure 6 shows the amino acid sequence of the heavy chain and light chain variable regions of Figure 6. The antibody 6G (including the Ch〇thia& Kabat CDR) i complementation determining region (CDR) portion is also shown. In another aspect, the invention comprises administering a 6G antibody variant having the amino acid sequence depicted in Table 8 In another aspect, the invention comprises administering an antibody comprising a fragment or region of antibody 6G or a variant thereof as shown in Table 8. In one embodiment, the fragment is the light chain of antibody 6G. In one embodiment, the fragment is a heavy chain of antibody 6g. In yet another embodiment, the fragment contains one or more variable heavy chains from antibody 6 (} and / or 129629.doc -) 4 200900079 heavy chain In yet another embodiment, the fragment contains - or a plurality of variable regions from the light and/or heavy chains not shown in Figure 8. In the yet-real slice, the light chain from antibody 6G is further contained. And/or a heavy chain - a plurality of CDRs. In another aspect, the invention comprises administering a polypeptide (which may or may not be an antibody) comprising the following Any one or more of: &) antibody (10) or one or more of its variants in Table 8; b) from antibody 6 (3 or the variants thereof shown in Table 8) CDR H3 of the chain; c) cdr L3 from the light chain of its variant shown in antibody (6) or Table 8; the three CDRs from the light chain of the antibody or its variant in Table 8; e) Three CDRs from the heavy chain of the variant shown in antibody 6G or Table 8; f) three cdr from the light chain of the variant shown in antibody 6G or in Table 8 and from antibody 6G or Table 8 Three cdr of the heavy chain of its variants shown. The invention further comprises a administering polypeptide (which may or may not be an antibody) comprising any one or more of the following: a) derived from an antibody One or more (one, two, three, four, five or six) CDRs of the variants thereof shown in 6G or Table 8; b) CDRs derived from the CDRs H3 from the heavy chain of antibody 6G; Or c) a CDR derived from the CDR L3 from the light chain of antibody 6G. In some embodiments, the CDR is the CDR shown in Figure 8. In some embodiments, derived from antibody 6G or as shown in Table 8. The variant Or the CDRs are at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least About 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and at least one of 6G or variants thereof, at least two, to 129629.doc •15·200900079 Less than three, at least four, at least five, or at least six cdr are consistent. In another sorrow, the invention comprises administering an antibody comprising a heavy chain variable region and a light chain variable region comprising a variable 6G heavy chain from the antibody set forth in SEQ ID NO:26 The three CDRs of the region, the light chain variable region comprising three CDRs from the antibody 6G light chain variable region set forth in SEQ ID NO:27. In another aspect, the invention comprises administering an antibody comprising a heavy chain variable region and a light bond variable region, wherein the heavy chain variable region comprises seq ID n〇: 28,

Three CDRs set forth in SEQ ID NO: 29.29 and SEQ ID NO: 30, the light chain cleavable region comprising the three of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33 CDR. In still another aspect, the invention comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 26 and a light chain variable comprising the amino acid sequence set forth in SEQ ID NO. Area. In still another aspect, the invention comprises the heavy bond amino acid sequence set forth in SEQ ID n: 361j and the light chain amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the CDR* Kabat CDRs. In other embodiments, the CDR is a Chothia CDR. In other embodiments, (10) is a combination with the Chothia CDR (also referred to as "combination cdr" or "extension cdr"). In other words, for any given embodiment containing more than one type of coffee, the CDR may be Kabat , Chothia and/or combination (10). In the second embodiment, the polypeptide (such as an antibody) comprises an amino acid sequence shown in SEq ID ,·5, wherein L1 is L, VW; Or W' where S3 is S, τ or G; where [4 is 卜r, a, v, s, 丁, 卩 or £ '· where V6 is V, 卜丁, P, C, Q, S, N or F; and its Yin Y7 129629.doc -16 - 200900079 sequence is the CDR3 in the heavy chain variable region. For the sake of convenience herein, in this context "is (lsy, or reference to amino acid) Refers to the position in π. ID to select the amino acid at a given position. For example, LV or I refers to the amino acid in the position of SEq ID N〇:5 [can be replaced by hydrazine or workmanship .

In some embodiments, a polypeptide (such as an antibody) comprises the amino acid sequence set forth in SEQ ID NO: 6, wherein Y8 is Y, A or hydrazine; and wherein A11 is A or S, and wherein K12 is K or A. In some embodiments, the amino acid sequence is CDR1 in the light chain variable region. In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence shown in SEQ ID NO: 8 wherein l^l, m, n, c, f, v, k, S, Q, G, Wherein G3 is G, s or τ; wherein D4 is butyl or s; wherein H5 is H:^L, wherein Y6 is γ, p, a, w, Q, M, S or E; wherein ¥, 匕, 〖, 11, 1 ', 八, ^, or 1^; and wherein 1^ is 1^, 1, T, S or V. In some embodiments, the amino acid sequence is CDR3 in the light chain variable region. In some embodiments, the polypeptide (such as an antibody) comprises a heavy chain variable region comprising: (4) a CDR1 region set forth in SEq ID N〇: 3; (b) SEQ ID NO: 4 a CDR2 region; and (c) a CDR3 region shown in SEQ m 5, wherein L4L, 乂 or 1; wherein γ^γ or w; wherein S3 is S, T or G; wherein L4 is L, R, A , V, S, T, Q4 Y, H, F, W, S, I, V^A 〇 129629.doc • 17· 200900079 In some embodiments, the polypeptide (such as an antibody) comprises a light chain variable region, The light chain variable region comprises: (4) a CDR1 region as shown in SEq ID N〇:6, wherein Y8 is Y, A or Η; and wherein A11 is A or s; and wherein 12 is 〖 or A '(b)SEQ ID n: the cdr2 region shown in 7; and (C) the CDR3 region shown in SEQ ID ,8, where Lul, m, N, C, F, V, K, S, Q, G, S' Wherein G3 is G, S or T; wherein D is 4 or S, wherein Η5 is Η or L; wherein Υ6 is γ, p, a, w, q, M, s or E; wherein V8 is V, L, K, H, T, A, mm; and wherein L, I, T, S or V. In some embodiments, the antibodies of the invention are human antibodies. In other embodiments, the antibody of the invention is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody (or polypeptide) is isolated. In some embodiments the 'antibody (or polypeptide) is substantially pure. The heavy chain region of the antibody can be derived from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgG1, IgG2, IgG3, and IgG4. In some embodiments, an antibody comprises a modified constant region, such as a constant region that is immunologically inert (which includes a portion that is immunologically inert and can be used interchangeably with the term having attenuating effector function), which, for example, does not trigger complement-mediated Lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC) or does not activate microglia. In some embodiments, the constant region is modified as described in Eur. L Immunol. (1999) 29:2613-2624; pCT Application No. PCT/GB99/01441; and/or British Patent Application No. 9809951 . In other embodiments the antibody comprises a human heavy 129629.doc -18-200900079 chain IgG2a constant region comprising the following mutations: A330P331 to S33〇S33l (refer to the amino acid number of the wild type lgG2a sequence). Eur. J.

Immunol· (1999) 29:2613-2624. In some embodiments, the antibody comprises a constant region of IgG4. The site contains the following mutations: E233F234L235 to P233V234A235. In still other embodiments, the constant 疋 region is deglycylated Sylated for N-linked glycosylation. In some embodiments, the constant region is directed against a N-linked sugar by mutating and/or flanking the oligosaccharide attachment residue (such as Asn297) as a residue of a portion of the N-glycosylation recognition sequence in the constant region Glycosylation. In some embodiments, the constant region is deglycosylated for N-linked glycosylation. The constant region is deglycosylated by N-linked glycosylation in an enzymatic manner or by expression in a glycosylation-deficient host cell.

In another aspect, the invention provides a polynucleotide (which can be isolated) which comprises a polynucleotide encoding a fragment or region of an antibody 9TL46G or a variable denier thereof as shown in Tables 3 and 8. . In one embodiment, the fragment is a light chain of antibody 9TL or 6G. In another embodiment, the fragment is a heavy chain of antibody oxime or (10). In yet another embodiment, the fragment contains one or more variable regions from the antibody 9TL or the light and/or heavy chain. In yet another embodiment, the fragment contains a sputum from antibody 9TL or 6G; one or more (also/or, three'four, five, six) complementarity determining regions (CDRs) of the 5^ strand and/or heavy chain ). [Embodiment] AMD's small gas model has been helpful to test the following hypothesis: lipid transport regulation abnormalities and class (4) deposition can promote the occurrence of steam plaque degeneration, glaucoma, diabetes, and sputum lesions (including macular edema) 129,629. The pathogenesis of retinal changes observed in doc -19- 200900079 and other related retinal degenerative diseases. Deposition has been extensively studied in Alzheimer's disease, and previous studies have shown that Aβ is associated with age-related macular degeneration (Y〇shida, τ. et al., L 〇f Clin·Invest·, 1 15(10): 2793_28〇 〇 (2〇〇5) ; Anders〇n, D, et al., Experimental Eye Research 78: 243_256 (2〇〇4);

Johnson, L. et al., PNAS, 99(18): 1 1820-1 1835 (2002)) and glaucoma (McKinnon SJ, Front Biosci 8: 1 140-56 (2003); Tatton et al., Surv Ophthalmol. 48: Potential role in S25_37 (2〇〇3)). However, there has been no discussion to date as to whether Αβ inhibitors can provide therapeutic benefit in treating macular degeneration by achieving retinal protection and/or recovery. In addition, there is no discussion as to whether any of the beta isoforms can differentially promote the pathogenesis of AMD. As discussed above, Αβ is the major component of neuritic plaques found in Alzheimer's disease. Αβ is a split product of a beta-based starch precursor protein (βΑρρ or Αρρ). ΑΡΡ is a type 1 transmembrane glycoprotein containing a large ectopic Ν terminal domain, a transmembrane domain, and a small cell Q 〇 end. Alternative splicing of a single Αρρ gene on chromosome 21 produces several isoforms of different numbers of amino acids: a type. Previous studies in Alzheimer's disease have read that (4) isoforms are essential for starch-like deposition, and APi42m. On the contrary, it can be a trigger molecule in the pathogenesis of Alzheimer's disease (McG〇wan, e. et al.

Ne_ 47: 191_199 _5)). Other studies in Alzheimer's disease have also shown that Αβ"0 isoforms can actually inhibit parenchyma deposition, and Αβ]-inhibitors can worsen the progression of Alzheimer's disease (Kim, j, et al. , NeUr〇bi〇logy of Disease, 27(3): (2)-Ink (2. 7)., 129629.doc -20· 200900079 The invention disclosed herein provides for administration of a therapeutically effective amount of an antibody 9TL or 6GS is a method for preventing and/or treating an ophthalmic disease in an individual, such as age-related macular degeneration (wet and dry), glaucoma, diabetic retinopathy (including Diabetic macular edema, Bruch's membrane rupture, myopia degeneration, ocular tumors, and other related retinal degenerative diseases. Antibody 9TL and its derivatives are described in WO 2006036291, the disclosure of which is incorporated by reference in its entirety. Herein, the antibodies and polypeptides used in the unexposed method bind to the c-terminus of Αβ]·4〇. Antibody 0G and its derivatives have been described in w〇2〇〇6〇36291 and w〇20061 18959, Reveal the content by reference to the full text The method of the present invention is intended to include all inhibitors of Aβ including, but not limited to, small molecule compounds and biological agents such as antibodies, antisense molecules, siRNA molecules, and ribonucleases. By way of explanation, otherwise the practice of the present invention will employ the well-known techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the art. These techniques are fully explained. Block the following documents: · Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (edited by RI·Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather 129629.doc -21-200900079 and PE, Roberts, 1998) Plenum Press ; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Gri Edited by ffiths and DG Newell, 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP· Calos, 1987); Current Protocols in Molecular Biology (F, M. Ausubel et al., ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al. Human Editing, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty. Edited, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Sheph.erd and C. Dean, ed. Oxford University Press, 2000); Using antibodies: a laboratory manual (E, Harlow and D· Lane ( Cold

Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and J.D. Capra' Harwood Academic Publishers, 1995) ° Definitions "Aβ peptide inhibitors" are any agents that reduce the production and/or deposition of Aβ peptides. Beta-like inhibitors include, but are not limited to, antibodies, antisense molecules, siRNA molecules, ribonucleases or small molecule compounds. Further, the Αβ peptide inhibitor is 129629.doc -22- 200900079 Any agent capable of binding Αβ peptide and reducing Αβ plaque deposition, including any agent capable of interrupting the cleavage of the starch-like precursor protein protein into the product Αβ peptide. Other targets that inhibit the production and deposition of Aβ peptide include, but are not limited to, for example, the ability to inhibit or suppress β-secretase (also known as BACE1 or memapsin_2) or a secretase complex (which is minimally composed of four individual proteins: Presenilin Small molecule therapeutics or siRNAs (presenilin), nicass (nicastHn), anterior pharyngeal defect (UApHd) and presenilin enhancer SCPENj)). An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc. via at least one antigen recognition site located in a variable region of an immunoglobulin molecule. . As used herein, the term "hai Hai terminology encompasses not only intact multi-strain or monoclonal antibodies, but also fragments thereof (such as Fab, Fab, F(ab,) 2, Fv), single-stranded (ScFv), mutants thereof, and A fusion protein of an antibody portion, and any other destructive configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include any class of antibodies such as IgG, IgA or igM (or a subclass thereof), and the antibody does not need to be genus (in any particular class. Depending on the antibody's amino acid sequence of its heavy chain, immunoglobulins Can be attributed to different categories. There are five main categories of immunoglobulins 'IgA, lgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as 坨 (1), IgG2, chemistry (7) , IgG4, IgAl, and IgA2. The heavy chain strange domains corresponding to different classes of immunoglobulins are called α, δ, ε, 7 and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. As used herein, "monoclonal antibody," refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., an individual antibody comprising the population may be naturally present in addition to a trace amount of 129629.doc -23-200900079. The mutations are all the same. Monoclonal antibodies are highly specific, targeting a single antigenic site, and, in contrast to multiple antibody preparations that typically include different antibodies to different determinants (antigenic determinants), each The antibody is directed against a single determinant on the antigen. The modifier "single plant" indicates that the antibody is characterized by a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, The monoclonal antibodies to be used in accordance with the present invention can be prepared by the fusion method described first by Kohler & Milstein, 1975, Nature, 256: 495, or by, for example, U.S. Patent No. 4,8 16,507. Monoclonal antibodies can also be isolated from phage libraries produced using techniques described in, for example, McCafferty et al, 199, Nature, 3 48: 5 52-5 54. As used herein, &quot "Humanized" anti-system refers to a non-human (eg, murine) antibody form that is a specific recombination immunoglobulin, immunoglobulin chain or fragment thereof (such as Fv, containing minimal sequence derived from a non-human immunoglobulin) Fab, Fab', F(ab')2 or other antigen-binding sequence of an antibody). To a large extent, a humanized antibody is one in which the residues from the complementarity determining regions (CDRs) of the recipient are from a non-human species. Human immunoglobulin (recipient antibody) substituted with a residue of cdr of a donor antibody, such as a mouse, rat or rabbit having the desired specificity, affinity and ability. In some cases The Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are neither found in the recipient antibody nor found in the input CDR or framework sequences. , but include it to further improve and optimize antibody potency. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, of which 129629.doc •24- 200900079 all or roughly _L all Non-human immunoglobulin

All FR regions in the body are human immunoglobulins - humanized antibodies will also preferably contain at least a portion of the immune (Fc)' which is typically the other of the human immunoglobulins. The antibody may have a modified Fe region as described in WO 99/58572. Other forms of humanized antibodies have one or more altered CDRs (-, two, five, six) relative to the original anti-system. They are also referred to as one or more of the " one or more from the original The cdR of the antibody (: £)11. As used herein, "human antibody" means an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human and/or having been manufactured using humans known in the art or disclosed herein. An antibody produced by any of the techniques of antibodies. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can be made using a variety of techniques known in the art. In one embodiment, the human anti-system is selected from a phage library, wherein the phage library exhibits human antibodies (Vaughan et al. '1996, Nature Biotechnology, 14: 309-314; Sheets et al, 1998, PNAS, (USA) 95: 61 57-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227: 381; Marks et al. '1991, J. Mol. Biol., 222: 581). Human antibodies can also be produced by introducing a human immunoglobulin locus into a transgenic animal (e.g., a mouse) in which the endogenous immunoglobulin gene has been partially or completely inactivated. The method of 129629.doc 25· 200900079 is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661, 〇16. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (this B lymphocytes can be recovered from the individual or can be immunized in vitro). See, for example, Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985); Boerner et al, 1991, J. Immunol., 147 (1): 86-95; and U.S. Patent No. 5,750,373 . As used herein, the terms n9TL" and, antibody 9TL" are used interchangeably to refer to antibodies produced by expression vectors having the accession numbers ATCC PTA-6124 and ATCC PTA-6125. The amino acid sequence of the heavy and light chain variable regions is shown in Figure 1. The CDR portions of antibody 9TL (including Chothia & Kabat CDRs) are graphically depicted in Figure 1. Polynucleotides encoding the heavy and light chain variable regions are shown in SEQ ID ΝΟ:9 and SEq ID:1〇. The nine-character characterization is described in the example. As used herein, the terms "6G" and "antibody 6G" are used interchangeably to mean having the heavy chain amino acid sequence set forth in SEQ ID NO: 36 and sEq4; Antibodies to the base acid sequence. The amino acid sequences of the heavy and light chain variable regions are shown in Figure 8. The CDR portions of antibody 6G (including Chothia and Kabat CDRs) are graphically depicted in Figure 8. The coding is heavy and light. The key polynucleic acid exhibits WSEQ ID excitation 38 and Qing m and: 39. The characterization of 6G is described in the examples. The term "polypeptide" '"oligopeptide", used to refer to an amino acid chain of any length The inclusion of a modified amino acid also encompasses polymers which have been naturally modified or borrowed from peptides and "proteins" interchangeable herein. The polymers may be linear or branched and may be heterozygous with non-amino acids. Modified amino acid polymer; 129629.doc -26 - 200900079 For example, disulfide bond & glycosylation, lipidation, acetate, lysine or any manipulation or modification, such as bonding to a labeled component. Included within the definition are, for example, one or more amino acid analogs (eg, including non-natural amine acid) Polypeptides, etc., and other modifications known in the art. It is understood that the polypeptides of the present invention are based on antibodies, so that the polypeptides may be in the form of single strands or related chains. As used herein, π polynucleosides are used interchangeably. Acid " or, nucleic acid" means any polymer of nucleotides of length f, and includes DNA and RNA. Nucleotides may be emulsified ribonucleotides, ribonucleotides, modified nucleotides Or a base and/or an analog thereof, or any substrate that can be incorporated into the polymer by DNA* RNA polymerase. The polynucleotide may comprise modified nucleotides, such as methylated nucleotides and An analog thereof. If present, the nucleotide structure can be modified before or after polymer assembly. The sequence of the nucleotide can be heterozygous with a non-nucleotide component. The polynuclear acid can be further modified after polymerization, such as Modifications by binding to labeled components. Other types of modifications include, for example, "cap", substitution of one or more of naturally occurring nucleotides with an analog, internucleotide modification, such as Modifications without a charge (eg methyl phosphonate, phosphorus) Acid triesters, phospholysates, urethanes, etc.) and modifications with charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), modifications with pendant moieties (such as proteins, such as nucleic acids) Enzymes, toxins, antibodies, Wei peptides, poly-L-lysine, etc.), modifications with intercalators (eg acridine, psoralen, etc.), modifications with chelating agents (eg metals, radioactive metals, boron) , oxidation metal, etc.), modification with an alkylating agent, modification with a modified bond (for example, α-helical heteromeric nucleic acid, etc.), and unmodified of a polynucleotide 129629.doc •27· 200900079 Form In addition, Any of the hydroxyl groups present in the sugar may be protected, for example, by a phosphonic acid group, a phosphate acid group, protected by a standard protecting group, or activated to prepare other bonds with other nucleotides, or may be associated with a solid support. Engage. 5, and 3, the terminal OH may be partially substituted with an amine or an organic capping group having a lji2_ carbon atom by acidification t. Other hydroxyl groups can also be derived to standard protecting groups. The polymorphic acid may also contain ribose or deoxyribose ribose in a similar form as commonly known in the art, including, for example, 2, 〇 _ methyl _, 2, s_ propyl, 2, qi - or 2'-azido-ribose, carbocyclic sugar analogue, alpha-spin isomer, sugar, such as arabinose, xylose or lyxose, pyranose, furanose, sedum A ketose (Sed〇heptulose), an acyclic analog, and an abasic nucleoside analog, such as a methyl riboside. One or more phosphodiester linkages may be replaced by an alternative linkage group. Such alternative linking groups include, but are not limited to, wherein the phosphate is via P(〇)S("thioester,), P(s)s("dithioester"), 〇)nr2("valerate"), P(0)R, p(〇)〇R, c〇 or cH2(,,甲甲

Examples of aldehyde ") substitutions wherein R*R| are each independently hydrazine or substituted or unsubstituted alkyl (1 〇 C C), aryl, Alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all bonds in a polynucleotide need to be the same. The foregoing description applies to all of the polynucleotides mentioned herein, including RNA and DNA. "variable region" of an antibody refers to a variable region of an antibody light chain, or a variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains are each composed of four framework regions (FR) joined by three complementary decision regions (CI) R, also referred to as hypervariable regions. The CDRs in each chain are! It is closely maintained and forms an antigen binding site for the antibody with CDRs from other chains. The existence of 129629.doc -28- 200900079 at least two techniques for determining CDRs: (1) methods based on cross species species variability (ie, Kabat et al., Sequences of Proteins of Immunological Interest, (Fifth Edition, 1991, National) Institutes of Health, Bethesda MD)); and (2) Methods for crystallographic studies based on antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). As used herein, CDR may refer to a CDR defined by either method or a combination of the two methods. The "constant region" of an antibody refers to the constant region of the antibody light chain or the antibody heavy chain, either alone or in combination. With antibodies or polypeptides, preferential binding " or "specific binding" The epitopes that are used interchangeably are well-understood terms in the art, and methods for determining this specificity or preferential binding are well known in the art. Reactions or associations with molecules and alternative cells or substances In contrast, if a molecule reacts or associates with a particular cell or substance more frequently, more rapidly, with greater duration and/or greater affinity, the molecule is referred to as "exhibition, specific binding".

Or "preferential binding" compared to the binding of an antibody to other substances, if the antibody is more 'affinity, steeper, more affinitive, and/or more consistent with the standard: 'anti-specific binding' Or, preferentially binds." Example: two with Αβι.4. The specific or preferential binding of the epitope is the ratio of the ratio, the Αβΐ' epitope or the non-αΡη. Affinity, easier, and/or greater continuation of this antigenic determinant (10) such as) and stem-dried special antibodies. Hunting should also be understood by reading this definition. E-binding antibody (or part or antigen-/, first stem-specific or preferential binding. Because of J29629.doc -29- 200900079, "specific binding" or "priority binding" is not required (Although it may include) exclusive combination. In general, '(but not - fixed) refers to the combination of meaning preferentially combined with 0 as used herein, "substantially pure" means at least 5〇% pure (ie no Contaminant), better at least 90% pure, better at least Pure, better to = pure, better, at least pure material. "Host cell" includes individual cells or cell cultures that may or have been recipients of vectors for incorporation into a polynucleotide insert. Cells include progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be identical to the original parent cell (in terms of morphology or genomic DNA complement). The progenitor cells include the polynuclears of the invention. The bitter acid is transfected in vivo. The term "Fc region" is used to define the c-terminal region of the immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. The border of the Fc region of the heavy chain can be altered 'but the human IgG heavy chain Fc region is usually defined as the amino acid residue from position Cys226 or from pr〇230 to its carboxy terminus. The number is the number of the EU index as in Kabat.

Kabat et al., Sequences of Proteins of Imunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically contains two constant domains: CH2 and CH3. As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Furthermore, preferred FcR is an FcR (y receptor) that binds to an IgG antibody and includes FcyRI, FcyRII, and 129629.doc -30-200900079

FcyRIII subclass receptors, including dual gene variants of these receptors and alternative splicing formats. FcyRII receptors include FcYRIIA ("activating receptors and FcyRIIB ("inhibiting receptors"), which have different similar amino acid sequences predominantly in their cytoplasmic domain. FcR lines are reviewed in Ravetcj^Kinet, 1991

Ann. Rev. Immunol·, 9:457-92; Capel et al., 1994

Immunomethods, 4:25-34; and de Haas et al, 1995, j. Lab Clin. Med., 126: 330-41. "FcR" also includes the neonatal receptor FcRn' which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, 1976, j. Immunol, 1 17: 587; and Kim et al, 1994, J. lmmunol, 24: 249). ° n Complement dependent cytotoxicity" and "CDC" means that the stem is lysed in the presence of complement. The complement activation pathway is initiated by binding the first component of the complement system (C1 q) to a molecule (e.g., an antibody) complexed with a homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al, J. Immunol. Methods, 202: 163 (1996). The π functional Fc region has at least one effector function of the native sequence Fc region. Exemplary "effector functions" include C1 q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity ( ADCC); phagocytosis; downregulation of cell surface receptors (eg, b cell receptors; BCR). Such effector functions generally require that the Fc region be combined with a binding domain (e. g., an antibody variable domain) and can be assessed using a variety of assays known in the art to assess the effect of such antibodies. "Native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. " variant Fc region, comprising acid modification due to at least one amine group 129629.doc-31 · 200900079 An amino acid sequence that differs from the native sequence by the amino acid sequence of the region 'but retains at least one effector function of the native sequence Fc region. The variant Fc region compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide Preferably, at least one amino acid substitution, such as from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions, is preferred in the native sequence Fc region or in the Fc region of the parent polypeptide. The medium variant Fc region will preferably have at least about 8 〇Q/❶ sequence identity to the native sequence region and/or to the Fc region of the parent polypeptide, and preferably has at least about 90% sequence identity to it, more preferably Having a sequence identity of at least about 95 Å, at least about 96%, at least about 97%, at least about 98%, at least about 99 Å/〇.

As used herein, "antibody-dependent cell-mediated cytotoxicity, and "ADCC" refers to a cell-mediated response in which non-specific cytotoxic cells (eg, natural killer (NK) cells) that express Fc receptors (FcR) are expressed. The neutrophils and macrophages recognize the binding antibodies on the target cells and subsequently cause lysis of the target cells. In vitro ADCC assays can be used to assess ADCC activity of the Z-bes of interest, such as those described in U.S. Patent No. 5,5,362, or 5,821,337. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) & NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model, such as the animal model disclosed in dynes et al., 1998, PNAS (USA), 95:652_656. As used herein, "effective amount I, or a pharmaceutical composition is an amount of a drug, a compound, or a desired result that is 'favorable or desired result or ''effective amount'' sufficient to achieve a beneficial use. Eliminate or reduce the risk, 129629.doc -32- 200900079 minus #2 serious privateness or delay the onset of the disease, the occurrence of chemical, group error and / red merger, the production of this disease; positive. The pathological phenotype presented during the development of the disease: for the therapeutic use: or the results include (but are not limited to) the partners m) saves or restores the visual network function or drug = sensitive The clinical result of the degree. The effective dose can be one or more traitors. For the purpose of the present invention, the effective dose of the drug, the chemical: the treatment of the Dan. u connect the ground to achieve preventive or therapeutic ', as in the clinical In the case, it is understood that the effective drug or pharmaceutical composition of the drug can be achieved with or without I, , compound, person, music, compound or pharmaceutical group. Therefore, in the investment - Can test the abuse of the ancient system In the case of a compound or a therapeutic agent, a combination of a community or a plurality of other agents may be achieved if the required, , or = fruit is achieved or has been achieved. 】T is considered as an early treatment with an effective amount as described herein, "treating" as a side of a miscellaneous fruit > ^ . ', ', favorable or desired result (including clinical, "fruit") . The beneficial or desired clinical outcomes for achieving the present invention include, but are not limited to, restoring and recognizing 10,000 times of retinal function. "The biological action of Αβ peptide" or, the role of knives in %, can be *β activity" means that in the eye disease, eight can be directly or indirectly for $, Β τ > ARA ^ ^, by function, Without being bound by theory, Αβ is involved in the regulation of abnormality in the delivery of 曰β. The indirect effects include (but not limited to) Αβ acting on retinal function and visual acuity. (4) Not limited to the use of “delay” The development of Zhanke disease means delaying the 'blocking, slowing, delaying, stabilizing and/or ΆI ^ ^ ^ I shoulder disease development. Depending on the history of the disease and or the individual of the alpha therapy, Xu ^, The delay can have a long-term meaning of change. For example, if you are familiar with this technology, you can see, enough or significantly delay 129629.doc -33 - 200900079 σ only covers prevention, because ^ ^ ^ ^ ^ ^ The disease development side, ... μ show far disease. The "delayed" eye h is a reduction in disease development rate within a given time = and/or a decrease in a given time frame when compared to not using the method. These comparisons are usually based on clinical studies using individuals with a significant number of controls. I. Development of ophthalmic diseases, 'meaning that the onset of an ophthalmic disease in an individual and/or the development of a standard clinical technique described herein that can detect the development of an ophthalmic disease also refers to the progression of the disease that may initially be undetectable. For the purpose of cost inventions, in this case, progression refers to a biological process such as a disease condition as determined by standard ophthalmology, or by a specialized test. Multiple diagnostic tests including (but not limited to) visual field, visual acuity, and fluoroscopy (7) reading

Angiography), electroretinogram, optical synchronous tomography (〇ct), visual evoked potential (VEP), indocyanine green, color vision, Amsler grid, intraocular pressure, and familiarity Other diagnostic tools known to the skilled person. Diagnostic tests for AMD include, but are not limited to, visual acuity, fundoscopic examination, fluorescein angiography, cyanocyanine and ocular synchronous tomography (OCT). "Development" includes appearance, recurrence, and seizure. As used herein, "onset" or "appearance" of an ophthalmic condition includes an initial onset and/or relapse. "protecting" retinal function as used herein refers to stabilizing or maintaining retinal function. Recovering retinal function as used herein refers to restoring retinal function after prior damage. The protection or recovery of retinal function can be determined by measuring statistically significant results (ie, p<〇.05), as measured by any of the above-mentioned diagnostic tools in the 129629.doc-34-200900079 section. , as / /, such as visual acuity, electroretinogram, visual field, fundus examination, fluorescent blood, π secluded surgery, indocyanine green and ocular synchronous tomography (OCT). For example, , as shown in Example 4 of _.., Ge F Wen, the retinal function of the system is shown by the recovery of the baud vibration 113⁄4 in the electroretinogram (ρ=〇. 〇〇 8). Visual acuity "maintaining" or "recovery" The "Tiantian" soap eye chart and a variety of ophthalmic diagnostic tools well known in the art are used. As used herein, "joint" Including simultaneous fun and/or at different times. Joint administration also covers ^ + cover, with the compound to be administered or as a separate composition. As used herein, the spleen machine # > a technical music means covering the individual with anti-Aβ antibody and another drug, which can be simultaneously and/or independently issued in this article, it should be understood that different doses or intervals can be administered. Anti-Aβ antibodies and other compounds. For example, anti-Aβ antibodies can be used, and other agents can be administered less frequently. It should be understood that administration of anti-Aβ antibodies and other drugs can be carried out by using (4) or different administration routes. Tincture 0 Bio 5 style " covers a variety of sample types that are self-contained and can be used for diagnostic or blood tests. This definition, blood and other liquid samples in stupid & ^, 〆1 biological sources , solid tissue sample, language 1 ^ tongue tissue test sample Or a tissue culture or derived therefrom, a cell and its progeny. The definition also includes a sample that can be made private after it has been obtained, such as by treatment with a reagent, dissolution, or a component (such as a protein or a polynucleus). The ridge & "~ Ganzi owe" is set or embedded in a solid or solid matrix to achieve the purpose of the slice. The term, biological sample|, covers clinical samples' and also includes cultures, cells, cell lysis, cell lysis, 129629.doc 200900079, serum, plasma, biological fluids, and tissue samples. "individual" (or "subject.^f") is a feeding animal, preferably a human. Feeding animals also include (but not limited to, things _, dog 1), $早::(4 As a cow, a sports animal, a pet, a primate, a mouse, and a rat. As used herein, a 'vector' means a construct that is capable of being delivered in a host cell and preferably capable of expressing _ Or a plurality of genes or sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked A or expression vectors, plastids, vesicular or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, vesicles A cargo or rib-eight expression vector encapsulated in a liposome, and certain eukaryotic cells, such as a producer cell. As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence can be a promoter or enhancer such as a constitutive or inducible promoter. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed. As used herein, "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, renders the ingredient biologically active and non-reactive to the individual's immune system. Examples include, but are not limited to, Any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of humectants. Preferred diluents for spray or parenteral administration are The acid salt buffer is used to buffer physiological saline or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known methods (for example, see corpse/mrmacewi/ca/ ☆z.ewce·?, 18th edition 'A. Gennaro, ed., Mack Publishing Co., Easton, PA, Ί 990 ', Anti-Remington, The Science and Practice of Pharmacy 129629.doc -36- 200900079 20th edition, Mack Publishing, 2000). The term "kon" as used herein refers to the association rate constant of an antibody to an antigen. The term "koff" as used herein is intended to mean the solution of an antibody from an antibody/antigen complex dissociation. The rate constant (off rate constant). As used herein, the term "KD" is intended to mean the equilibrium dissociation constant of antibody-antigen interactions. Methods of Making Compositions and Compositions Anti-Aβ Antibodies and Polypeptides: I. Antibody 9TL and 9TL Derived antibodies and peptides

The present invention encompasses a composition comprising a pharmaceutical composition comprising an antibody 9TL and variants thereof as shown in Table 3 or a polypeptide derived from the antibody 9TL and variants thereof shown in Table 3; and comprising a 9TL antibody and Change

The sequence of polynuclear «. As used herein, a composition comprising an antibody or polymorphism (which may or may not be an antibody) that binds to the Αβι.40 Κ terminus (and/or- or a plurality of antibodies encoding one or more antibodies that bind to the Api^c terminus) Or a polymorphic acid sequence of a polypeptide. Such compositions may additionally comprise humans, excipients, such as pharmaceutically acceptable excipients, including buffering agents: which are well known in the art. And the polypeptide is characterized by any one of the following features (-m(4) and Αβ1, the c-terminal peptide 28, but not significantly or from (4) nine; (8) and the ", inhibition occurs in the individual _ Powder broad peptide 33·4. Combination, · (4) plaque; (stick prevention, change Γ two (:) species salty less (four) ^. Second eve ophthalmic disease symptoms, the ophthalmology 129629.doc 37 200900079 disease including (Ren Limited to age-related macular degeneration (dry and wet), glaucoma, diabetic visual lesions (including macular edema) and other related visual, decidual degeneration), (7) significant protection or recovery of retinal function; (g) significant retention of visual acuity or The antibodies and polypeptides of the invention may also exhibit a desired safety profile as compared to other reported anti-Aβ antibodies. Accordingly, the present invention provides any of the following, or any of the following Composition (including pharmaceutical composition): (hook antibody 9 sputum or its variants in Table 3; (b) antibody 9TL or a fragment or region thereof of the variant shown in Table 3; (4) antibody 9TL Or the light chain of the variant thereof shown in Table 3; (d) the heavy chain of the antibody 9TL or the variant thereof shown in Table 3; (about the light from the antibody 9TL or the variant thereof shown in Table 3) One or more variable regions of the chain and/or heavy chain; (f) one or more CDRs (-, two, three, four, five or six CDRs of the antibody 9TL or variants thereof shown in Table 3) (g) CDR H3 from the heavy chain of antibody 9TL; (h) CDR L3 from the light chain of the antibody 9TL or its variant shown in Table 3; (1) from antibody 9TL or as shown in Table 3 Three CDRs of the light chain of the variant; (1) three CDRs from the heavy chain of the variant shown in antibody 9tl or Table 3; (k) from the variants of antibody 9TL or Table 3 The three CDRs of the chain and the three CDRs of the heavy chain from the antibody 9TL or the variant thereof shown in Table 3; and (1) the antibody comprising any of (b) to (k). The invention also provides Polypeptide of any one or more of the above. The CDR_portion of antibody 9TL (including Chothia and Kabat CDRs) is graphically depicted in Figure 1. The cdR region is well within the art. Understanding 129629.doc -38- 200900079 In some embodiments, the CDR can be a combination of 讣 and chothia CDRs (also known as a combined CDR" or "extended CDR"). In some embodiments, the CDR is a Kabat CDR. In other embodiments, €1)11 is a Ch〇thia CDR. In other words, in embodiments having more than one CDR, the CDR can be, Chothia, a combination, or any combination thereof.

In some embodiments, the invention provides a polypeptide (which may or may not be an antibody) comprising at least one CDR, at least two, at least three, substantially identical to the 9TL or the variant thereof shown in Table 3, At least four, at least five or all six CDRs are at least one CDR, at least two, at least two or at least four, at least five or all six CDRs. Other embodiments include having at least two, three, four, five or six CDRs substantially identical to 9TL or derived from at least two, three, four, five or six CDRs of the 9TL antibody. In some embodiments, the at least one, two, three, four, five or six (: 1:) 11 and 9 Ίχ or at least one, two, three of its variants shown in Table 3 At least 85%, 86%, 87%, 88%, 89%, 90%, 95%/〇' 960/〇, 97%, 98% or 99% . It will be appreciated that for the purposes of the present invention, although the degree of activity may vary (may be larger or smaller) than the variants shown in 9TL or Table 3, the binding specificity and/or overall activity is generally maintained. The invention also provides a polypeptide (which may or may not be an antibody) comprising an amino acid sequence of a variant thereof as shown in 9TL or Table 3, the amino acid sequence having any of the following : 9TL or at least 5 contiguous amino acids of the sequence of variants thereof shown in Table 3, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least About 2 邻 129 129.doc •39· 200900079 Amino acid, at least about 25 contiguous amino acids, at least about contiguous amines ^', at least 3 of its t« amino acids are from 9tl (Figure The variable region of the variant shown by υ or 艸. In the embodiment, the variable region is derived from the light chain of 9TL. In another embodiment, the variable region is derived from the heavy chain of 9tl. The polypeptide has contiguous amino acids (lengths described above) from the heavy and light chain variable regions of 9TL. In another embodiment, five (or more than five) contiguous amino acids are from the figure shown in Figure! The complementarity determining region (cdr) of 9TL. In some embodiments, the adjacent amino acid is derived from the variable region of 9τχ. II. Antibody 6G and 6G derivatives Antibodies and Polypeptides The invention further provides a method of treating an ophthalmic disease comprising administering an antibody or polypeptide that binds to Αβ^ο, Αβ__42 and Αβυ3. In some embodiments, the antibody or polypeptide is compared to Αβι·42 and Ah3 binds to a higher affinity for binding to Αβ^. In some embodiments, the antibody binds to Αβΐ36, Αβ!-38, and Αβ] _39. In some embodiments, the antibody binds. In some embodiments, The antibody binds to gossip. In some embodiments, the antibody or polypeptide binds to an epitope comprising amino acid 25-34 and 40. The present invention also provides a method of treating an ophthalmic disease, It comprises administering a pharmaceutical composition comprising any one of the antibodies or polypeptides described herein (such as antibody 0G and variants thereof shown in Table 8 or derived from antibody 6g and in Table 8) Polypeptides as shown herein; or as described herein, a composition comprising one or more antibodies or polypeptides that bind to the C-terminus of Αβ^ (which may or may not be antibodies) And/or one or more contain One or more of the antibodies or polypeptides that bind to the c-terminus of the eight (9), the sequence of 129629.doc -40-200900079. These compositions can be used to form A &gt; aa 匕3 σ suitable sputum Excipients such as those disclosed in the medical department include well-known. In the art, the antibodies and polypeptides of the invention are characterized by any one or more of the following characteristics: (a) Αβ λ 〇Αβ, and Αβ, .43 bind; _Ak, UAPi.43 bind, _ and |4. The affinity of the binding is higher than the affinity of the virtual 38 AU Αβΐ_43 binding, (4) binding to the epitope of Αβ! including amino acid 25-34 and 40; (4) binding with I, lattice ^ and Αβ卜39' But with the parallel disk ar +44~ people! _ _ 40,, 'mouth δ has lower affinity: (4) with less than about 1 - heart combined with shirt (10); _β22 · 35 combination: (8) and Αβ28·4. (8) does not bind to Αρρ expressed in cells; (1) reduces acne spots on the eye of an individual; (1) treats, prevents, and ameliorates symptoms of one or more ophthalmic diseases including, but not limited to, age-related only Plaque degeneration (dry and wet), glaucoma, diabetic retinopathy (including macular edema) and other related retinal degenerative diseases; (9) significant protection or recovery of retinal function; and (1) significant retention or recovery of visual acuity . The antibodies and polymorphisms of the invention may also have the attenuating effect functions described herein. Antibodies and polypeptides with impaired effect functions can exhibit a desired safety profile compared to other reported anti-Ap antibodies. For example, the compositions of the present invention may not cause any one or more of the following: significant or unacceptable levels of bleeding in the vascular structure of the brain (cerebral hemorrhage); meningoencephalitis (including magnetic resonance scanning) Increased white blood cell count in cerebrospinal fluid; inflammation of the central nervous system. Accordingly, the present invention provides any of the following, or a composition (including a pharmaceutical composition) comprising any of the following 129629.doc 41 200900079: (a) antibody 6g or shown in Table 8 a variant thereof; (b) a fragment or region of the antibody 6G or the variant thereof shown in Table 8; '(c) antibody 6G or the light chain of the variant thereof shown in Table 8, (d) antibody 6G Or the heavy chain of the variant thereof shown in Table 8; (e) one or more variable regions of the light chain and/or heavy chain from the antibody 6G or the variant thereof shown in Table 8; (1) Antibody 6G Or one or more CDRs (-, two, three, four, five or six CDRs) of the variants thereof shown in Table 8; (g) CDR H3 from the heavy chain of antibody (10); (h) from antibody 6G or CDR L3 of the light chain of its variant shown in Table 8; (1) three CDRs of the light chain from the antibody 6G or the variant thereof shown in Table 8; (1) from antibody 6 (} or Table 8 Three CDRs of the heavy chain of its variant are shown; (k) three CDRs from the light chain of antibody 6 (3 or its variant shown in Table 8 and from antibody 6G or as shown in Table 8 The three CDRs of the heavy chain of its variant; and (1) The antibody of any one of (b) to (k). The invention also provides a polypeptide comprising any one or more of the above. The antibody 6G (including the Chothia & Kabat CDR) is illustrated diagrammatically in Figure 8. The CDR portion. The determination of the CDR regions is well within the skill of the art. In some embodiments, the invention provides a polypeptide (which may or may not be an antibody) comprising substantially as shown in 6G or in Table 8. At least one CDR, at least two, at least three, at least four, at least five or all six CDRs of the variant - at least one CDR, at least two, at least three or at least four, at least five or All six CDRs. Other embodiments include at least two, three, four, five or with at least two, three, four, five or six CDRs of 6G or derived from 6G Six 129629.doc -42 · 200900079 CDR antibodies. In some embodiments, the at least one, two, three, four, five or six CDRs are mutated with 6G or At least one, two, three, four, five or six CDRs of the body are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99%. It should be understood that for the purposes of the present invention, although the degree of activity is 6G or Table 8 The variants shown therein may vary (may be larger or smaller) but generally retain binding specificity and/or overall activity. The invention also provides a polypeptide (which may or may not be an antibody) comprising 6A or the amino acid sequence of the variant thereof shown in Table 8, the amino acid sequence having any of the following: 6G or at least 5 contigs of the sequence of the variant thereof shown in Table 8 An amino acid, at least 8 contiguous amino acids, at least about 1 contiguous amino acid, at least about 15 contiguous amino acids, at least about 2 contiguous amino acids, at least about 25 contiguous amino acids, At least about 3 adjacent amino acids, wherein at least 3 of the amino acids are from 6G (the variable regions of the variants thereof shown in Figure 8 or Table 8). In one embodiment, the variable region is from a 6G light chain. In another embodiment, the variable region is derived from the heavy chain of the yang. An exemplary polypeptide has a contiguous amino acid (length as described above) from the heavy and light chain variable regions of 6G. In another embodiment, five (or more than five) contiguous amino acids are derived from the 6G complementarity determining regions (CDRs) shown in Figure 8. In some embodiments, the adjacent amino acid is derived from the variable region. As exemplified in the Examples below, the binding affinity of the antibodies and polypeptides of the invention may vary and need not be, but may be, a particular value or range. The binding affinity (KD) of the antibodies and polypeptides of the present invention to the Αβ peptide (including or Αβ&quot;, Ah or peptide) may be from about 0·10 ηΜ to about 〇8〇ηΜ, about 5 129629.doc -43. 200900079 nM to about 0.75 nM and about 0.18 nM to about 0.72 nM. In some embodiments, the binding affinity is about 2 ρ Μ, about 5 ΡΜ, about 1 〇 ρ Μ, about 15 ρ Μ, about 20 ΡΜ, about 40 ρ Μ or greater than about 40 ρ 。. In one embodiment, the binding affinity is between about 2 ρ Μ and 22 ρ 。. In other embodiments, the 'binding affinity is less than about 丨〇ηΜ, about 5 ηΜ, about i ηΜ, about 9〇〇ΡΜ, about 8GQ ρΜ, about ρΜ, about 6GG ΡΜ, about 5GG ρΜ, about 400 ρ Μ, ^ π λ , 'woo ΡΜ , about 200 ΡΜ , about 150 ρ Μ , about 1 〇〇ΡΜ , about 90 ρ Μ , δ η , , θ 80 ρ Μ , about 70 ρ Μ , about 60 ρ Μ , about 50 ρ Μ , about 40 ΡΜ , about 10 ρΜ. In some embodiments, the binding affinity is about 10 nM. In other embodiments, the binding affinity is less than about 丨〇nM, less than about 50 nM, less than about 100 nM, less than about 150 nM, less than about 200 nM, less than, minus about 250 nM, less than about 500 nM or less than about 1000 nM. In each of the other examples, the 'binding affinity is less than about 5 nM. In the straight embodiment, the 〜8, 犯~& affinity is less than about 1 nM. In other implementations In an example, the affinities of the junction are about affinities j or 〇.07. In other embodiments, in the knot, 辜/about ο.1 secret or less than about 0.07 nM. In other embodiments PM, about coffee and sex are about 1 〇ΠΜ, about 5 nM, about 1 nM, about _ 400 PM, about 3 PM, about 7 〇 0 PM, about 600 PM, about 5 〇〇 PM, about 90 PM, about 〇 〇PM, about 2 〇 0 PM, about 150 PM, about 100 PM, PM, about 30 λ ΓΡΜ, about 7 〇 PM, about 6 〇 PM, about 5 〇 pM, about 40 about 1 〇 pM, P / 'about 1 Any one of 〇_ to about 2 PM, about 5 pM, - some of the examples, 15 PM, about 2 〇 PM, or about 4 〇 PM. In combination with about 9 〇〇 λ ' Affinity is about 10 hearts, about 5 nM, about i P 'about 800 pM, about 7 〇〇pM, about 6 〇〇 _, about 129629.doc -44 - 200900079 500 pM, about 4 GG pM, about 3 GG pM, about 2 GG pM, about ι5 〇 μ about 100 PM, about 90 pM, about 80 pM, about 70 pM, about 6 〇pM, about 5 〇 PM, about 40 pM, about 30 PM, about any of i〇pM. In his embodiment, the binding affinity is about 2 , about 5 pM, about pM', about 15 PM, about 20 pM, about 40 pM or more than about 40 PM. The antibody of the present invention or poly R can be associated with Αβ,, Αβΐ42 and/or Αβι·42 peptides. In one embodiment, the antibody, the polypeptide or the polypeptide is combined with at least Αβι, °, 、, 。. Ρΐ-42 peptides The antibodies and polypeptides of the invention may also be associated with Αβι·36, Ah ”, Ah Αβ丨-39, Any one or more of Αβ〗 _42 and ApK43 are combined, but in this embodiment, the binding affinity of any one or more of these peptides is small, and the binding affinity for Αβ]. In some embodiments, the antibody or more; and Αβ, .36, Αβυ?, Αβΐ 38, Αρΐ 39, α (3ι_42 and Αβ peptide j τ < any one or the evening Kd is the KD of Αβ丨_40 At least about 5 times, less than about 10 times, at least about 20 times, at least about 3 times, at least about 4 times, about 50 times less, at least about 80 times, at least about 1 times, at least about 15 〇, at least about 200 or at least about 250. The invention also provides methods of making any of such antibodies or polypeptides. The antibodies of the invention can be made by procedures known in the art. It can be produced by proteolysis or other degradation of antibodies, by recombinant methods as described above (ie, single or fusion polypeptides) or by chemical synthesis. Polypeptides of antibodies can be conveniently prepared by chemical synthesis, especially up to about 5. The shorter amino acid of the amino acid. Chemical synthesis methods are known in the art and are commercially available. For example, an automated peptide synthesizer using a solid phase method can be 129629.doc •45- 200900079 Manufacture of antibodies. See also U.S. Patent No. 5,807,715; 4,816,5 No. 67; and 6,331, 415. In another alternative, the antibody can be produced recombinantly using procedures well known in the art. In one embodiment, the polynucleotide comprises a heavy chain encoding the antibody and/or Sequence of a light chain variable region. In another embodiment, a polynucleotide comprising a nucleotide sequence is cloned into one or more vectors for expression or propagation. Sequences encoding the antibody of interest can be The vector is maintained in a host cell and the host cell can then be expanded and cryopreserved for later use. The vector (including the expression vector) and the host cell are further described herein. The invention also encompasses antibodies of the invention (such as 9TL and 6G) A single-chain variable region fragment (&quot;scFv&quot;). A single-chain variable region fragment is made by ligating a light chain and/or heavy chain variable region using a short linker peptide. Bird et al. (1988) Science 242: 423-426. An example of a linker peptide is (GGGGS) 3, between the slow-acting terminus of the bridge-variable region and the amine-based terminus of the other variable region, about 3. 5 Please. Others have been designed and used. Sequence linker. (1) Postman (test). Linker can be modified For other functions, such as drug attachment or attachment to a solid support. Single-stranded variants can be made recombinantly or synthetically. For the synthetic production of scFv, an automated synthesizer can be used. For the recombinant production of SCF" can contain coded scFv The appropriate plastid of the polynucleus (IV) is introduced into a suitable host cell, which may be true (d), such as a yeast, plant, insect or mammalian cell; or a prokaryotic 'such as a large bowel taste (3) can be encoded by, for example, poly The conventional operation of the acid-ligation reaction is carried out. The resulting scFv can be isolated using standard protein purification techniques known in the art. 129629.doc -46 - 200900079 Other forms of single chain antibodies, such as bifunctional antibodies, are also contemplated. A bifunctional antibody is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single peptide chain, but are used in a linkage that is too short to allow pairing between the two domains on the same chain. Forcing the domains to pair with the complementary domain of another chain and creating two antigen binding sites (see, for example, H〇iHger, p. et al. (1 outside 3)

Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J. et al. (1994) Structure 2: 1121-1123). For example, antibodies disclosed herein can be used to prepare bispecific antibodies (monoclonal antibodies having binding specificities for at least two different antigens (i.e., Αβΐ 4〇 and Api 42)). Methods for making bispecific antibodies are known in the art (e.g., in Suresh et al., 1986, Methods in Enzym. 1 〇 121: 210). Traditionally, recombinantly produced bispecific anti-systems have been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, two of which have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539). . According to a method for producing a bispecific antibody, an antibody variable domain having a desired binding specificity (antibody/antigen combining site) is fused to an immunoglobulin constant domain sequence. Preferably, it is fused to an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 &amp; CH3 region. It preferably has a first heavy chain region (CH1) containing a site necessary for light chain binding (present in at least one of the fusions). The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. In the examples where the ratio of the three polypeptide bonds used in the construction is: the ratio provides the optimum yield, this provides a high degree of flexibility in adjusting the mutual ratio of the three polypeptide fragments. However, when at least two of the equal ratios of 129,629.doc •47-200900079 peptide chains produce high yields or when the ratios are not of particular importance, it is possible to insert two or all three polypeptide bonds in the expression vector. The coding sequence. In one method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in the arm, ^ ^ Λ , ^ sighing a heterozygous immunoglobulin heavy chain in the other arm _ Light chain pair (providing a second binding specificity). This asymmetric structure with an immunoglobulin light chain in only the half-double-specific molecule promotes the separation of the bispecific compound from the undesired and optically combined globulin chain. This method is described in PCT Public Relations, et al., WO 94/04690, published March 3, 1994. Heterologous conjugated antibodies comprising two covalently linked antibodies are also within the scope of the invention. Such antibodies have been used to target immune system cells to undesired cells (U.S. Patent No. 4,676,980, bra, 唬) and for the treatment of HIV infection (PCT Application Publication No. W/91/00360 and WO 92/200373; EP with 9). ° Use a heterozygous parental approach to make heterologous conjugated antibodies. Suitable cross-linking

Agents and techniques are well known in the art and are described in U.S. Patent No. 4,676,980. Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, an immunotoxin can be constructed using "sputum change reaction or by formation of a thioether bond. Examples of suitable 丨na to for this purpose include iminothiolate and thiol-4. - mercaptobutyric acid imine g. This technology can be used φ

T Any method known to produce humanized 129629.doc-48-200900079 antibody comprising one or more CDRs of antibody 9TL, 4 bamboo derived from one or more CDRs of antibody 9TL. For example, four general steps can be used to humanize a monoclonal antibody. The steps are: (1) determining the nucleotides of the starting antibody light and heavy chain variable domains and predicting the amino acid sequence, and (2) designing the humanized antibody, ie determining which to use during the humanization process. Antibody framework regions, (3) actual humanization methods/technologies, and (4) transfection and performance of humanized antibodies. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; No. 6,548,640. In recombinant humanized antibodies, the Fc portion can be modified to avoid interaction with the Fcy receptor and the complement immune system. This type of modification is made by Cambridge

The technique for preparing such antibodies is described in Dr. Mike Clark, Department of Pathology, University, and in WO 99/58572, published on Nov. 18, 1989. For example, if antibodies are used in clinical trials and treatments in humans, the constant regions can be designed to be more similar to human constant regions to avoid an immune response. See, for example, U.S. Patent Nos. 5,997,867 and 5,866,692. The present invention encompasses modifications to antibodies 9TL and 6G, including functionally equivalent antibodies that do not significantly affect their properties, and variants that enhance or decrease activity and/or affinity. For example, the amino acid sequence of antibody 9TL or 6G can be mutated to obtain an antibody having the desired binding affinity for the target Αβ peptide. Polypeptide modifications are routine practice in the art and need not be described in detail herein. Examples of polypeptide modifications are shown in the examples. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, which do not significantly adversely alter functional activity. 129629.doc -49-200900079 One or more deletions or additions of amino acids or use of chemical analogs . Amino acid sequence insertions include sequences of amino-terminal and/or carboxyl-terminal fusions and single or multiple amino acid residues ranging from one residue to a polypeptide containing one hundred or more residues in length insert. Examples of the terminal insertion include an antibody having an N-terminal thiol sulfhydryl residue or an antibody fused to an epitope tag. Other insertion variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide which increases the serum half-life of the antibody. The substitution variant has at least one amino acid residue in the removed antibody molecule and a different residue inserted into its position. Although the most interesting sites for substitution mutation induction include hypervariable regions, FR alterations are also covered. The conservative substitutions are shown in Table 1 under the heading "Conservative Substitution". If such substitutions result in a change in biological activity, a more substantial change, as described in Table 1 below, as an &quot;exemplary substitution n&quot; or as further described below with reference to an amino acid, can be introduced and the product screened. Table 1: Amino acid substitutions Original residues Conservative substitutions Exemplary substitutions Ala (A) Val Val ; Leu ; lie Arg ( R ) Lys Lys ; Gin ; Asn Asn ( N ) Gin Gin ; His ; Asp , Lys ; Arg Asp (D) Glu Glu ; Asn Cys ( c ) Ser Ser ; Ala Gln ( Q ) Asn Asn ; Glu Glu ( E ) Asp Asp ; Gin Gly ( 6 ) Ala Ala His ( H ) Arg Asn ; Gin ; Lys ; Arg lie (I Leu Leu ; Val ; Met ; Ala ; Phe ; Leucine Leu ( L ) lie leucine ; lie ; Val ; Met ; Ala ; Phe 129629.doc -50- 200900079 Original residue conservative substitution exemplification Substituting Lys(K) Arg Arg ; Gin ; Asn Met ( Μ ) Leu Leu ; Phe ; lie Phe (F) Tyr Leu ; Val ; lie ; Ala ; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T Ser Ser Trp (W) Tyr Tyr ; Phe Tyr ( 7 ) Phe Trp ; Phe ; Thr ; Ser Val ( V ) Leu lie ; Leu ; Met ; Phe ; Ala ; Different substitutions to achieve substantial modification of the biological properties of the antibody: (a) the polypeptide backbone structure within the substitution region, eg, in a sheet or spiral configuration, and (b) at the target site The charge or hydrophobicity, or (c) bulk of the side chain. Naturally occurring residues are grouped based on common side chain properties: (1) Non-polar: positive leucine, Met, Ala, Val, Leu, lie; (2) Uncharged polarity: Cys, Ser, Thr, Asn (3) Acidic (negatively charged): Asp, Glu; (4) Authentic (positively charged): Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. Non-conservative substitutions are achieved by exchanging members of one of these categories into another. Any cysteine residue that does not involve maintaining the proper configuration of the antibody can generally also be substituted with a serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, a cysteine bond can be added to the antibody to improve its stability, especially when the antibody is an antibody fragment such as an Fv fragment. Amino acid modifications can vary between modifying or modifying one or more amino acids to a full weight 129629.doc • 51 · 200900079 designing regions such as variable regions. Changes in the variable regions can alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are achieved within the CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are achieved in the CDR domain. In still other embodiments, the CDR domain is CDR H3 and/or CDR L3. Modifications also include glycosylated and non-glycosylated polypeptides as well as polypeptides having other post-translational modifications such as glycosylation, acetylation and phosphorylation with different sugars. The anti-system is glycosylated at conserved positions in its constant region (Jefferis and Lund, 1997, Chem. Immunol. 65:1 1-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chain of immunoglobulin affects the function of proteins (Boyd et al, 1996, Mol. Immunol. 32: 1311-1318; Wittwe and Howard, 1990, Biochem. 29: 4 175-4 180) and in the glycoprotein fraction Intramolecular interactions (which can affect the conformation of glycoproteins and the three-dimensional surface presented) (Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on a specific recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cell modification with tetracycline-regulated expression of β(1,4)-fluorenylglucosyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of GlcNAc, has been reported. ADCC activity (Umana et al, 1999, Mature Biotech. 17: 176-180). The glycosylation of antibodies is usually either N-linked or sputum-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue. Tripeptide sequence aspartic acid-X-serine, aspartic acid-X-threonine and aspartate 129629.doc -52- 200900079 Ampoule cysteamine (where x is in addition to guanamine Any amino acid other than acid is used; AX δ 邛 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Thus, the presence of any of these tripeptide sequences in a polypeptide produces a potential glycosylation site. 〇_Linked glycosylation means one of glycosyl galactosamine, half sugar or xylose attached to a hydroxyl amino acid, most commonly seric acid or threonine It is possible to use 5-hydrazinoic acid or 5-hydrazinoic acid. Addition of a glycosylation site to the antibody (for an N-linked glycosylation site) is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences. The alteration can also be achieved by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting one or more serine or threonine residues (for a 0-linked glycosyl group) Chemical site). The glycosylation pattern of the antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Although cell types are used to express recombinant glycoproteins (eg, antibodies), glycosylation patterns of antibodies can be expected to change as potential therapeutic agents are rarely natural cells (see, for example, Hse et al., 1997, J Bi〇1. Chem) 272: 9062-9070). In addition to host cell selection, factors that affect glycosylation during antibody recombinant production include growth patterns, media formulations, culture densities, oxygenation, pH, purification protocols, and the like. Various methods have been proposed to alter the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (U.S. Patent No. 5, 〇 47, 3 35; No. 5, 510, 261) And No. 5, 278, 299). It can be enzymatically derived from sugars 129629.doc -53 - 200900079 Protein_removing glycosylation or some #feeding glycosylation, for example using endosaccharide f. H), as described in Example 3, N-glycosidase F, endo-saccharide: endoglycosidase F2, endo-glycosidase F3 to remove. In addition, recombinant primary cells can be genetically transformed into 丄 °. X. These and similar techniques are well known in the art for processing certain types of polysaccharides. G-decoration methods include the use of coupling techniques known in the art including, but not limited to, enzymatic means, oxidative substitution, and chelation. The repair mark can be used, for example, for the plague &amp; The modified 9 polypeptides are prepared using established procedures in the art and can be screened using standard assays known in the art, which are described below and in the Examples. In some embodiments of the invention, the antibody comprises a modified constant region, such as an immunologically inert or partially inert constant region, for example, does not trigger complement-mediated cytotoxicity, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADcc) Or does not activate microglial cells; or has reduced activity in any one or more of the following (compared to unmodified antibodies): triggers complement-mediated lysis, stimulates antibody-dependent cell-mediated cells Toxicity (ADCC) or activation of microglial cells. Different modifications of the constant region can be used to achieve a level and/or combination of effector functions. See, for example, Morgan et al., /mmwno/ogy 86:319-324 (1995); Lund et al., /. /mmw (iv) 157:4963-9 157:4963-4969 (1996); Idusogie et al., J. /mwM«o /og_y 164:4178-4184 (2000) ; Tao et al, J. 143: 2595-2601 (1989); and Jefferis et al., and eWews 163:59-76 (1998). In some embodiments, such as Eur. J. Immunol. (1999) 29: 2613-2624; PCT Application No. 129629.doc 54. 200900079 PCT/GB99/01441; and/or UK Patent Application No. 98〇9951 The constant region was modified as described in No. 8. In other embodiments, the antibody comprises a human heavy chain IgG2a constant region comprising the following mutations: 1 to S33 〇 S3; 31 (refer to the amino acid number of the wild type IgG2a sequence). Eur. j.

Immunol. (1999) 29:2613-2624. In still other embodiments, the constant region is deglycosylated for N-linked glycosylation. In some embodiments, the constant region is directed against N-linked glycosylation by mutating or flanking a glycosylated amino acid residue as a residue of a portion of the N-glycosylation recognition sequence in the constant region Glycosylation. For example, the N-glycosylation site N297 can be mutated to A, Q, K or Η. See Tao et al, J. Fantasy; 143: 2595-2601 (1989); and Jefferis et al, 163: 59-76 (1998) In some embodiments, the constant region is deglycosylated for n-linked glycosylation The cleavage region can be deglycosylated for N-linked glycosylation by enzymatic means (such as removal of carbohydrates by enzyme PNGase) or by expression in a glycosylation-deficient host cell. Antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572, published November 18, 1999. In addition to binding domains to the target molecules, such antibodies comprise substantially immunologically immunized with humans. The effector domain of all or part of the homologous amino acid sequence of the constant domain of the globulin heavy chain. These antibodies are capable of binding to the target molecule 'without triggering significant complement-dependent lysis' or target cell-mediated destruction. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or FcyRIIb. These are typically based on a fusion domain derived from two or more human immunoglobulin heavy chain CH2 domains. Especially suitable for chronic antibody therapy to avoid 1 29629.doc -55- 200900079 Inflammatory and other adverse reactions to conventional antibody therapies. The invention includes affinity matured embodiments. For example, affinity matured antibodies can be made by procedures known in the art ( Marks et al, 1992, Bio/Technology, 10: 779-783; Barbas et al, 1994, Proc Nat. Acad. Sci, USA 91: 3809-38 13; Schier et al, 1995, Gene, 169: 147-155 Yehon et al., 1995, j. Immunol., 155: 1994-2004; Jackson et al. '1995, j Immun〇1, 154(7): 3310-9 'Hawthorns et al.' 1992, j. m〇1. Biol., 226:889-896; and WO 2004/058184). The following methods can be used to modulate the affinity of an antibody and to characterize cdr. A CDR that characterizes an antibody and/or alters (such as a modification) a polypeptide such as an antibody The method of binding affinity is called "Hbrary scanning mutagenesis". 1. In general, library scanning mutation induces the following work. Using one of the methods identified in the art, one or more amino acid positions in the CDR Two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid substitutions. Thereby producing a small pure line library (in some embodiments, one for each analyzed amine based position), each having a complexity of two or more members (if two or two) The above amino acid is substituted at each position). In general, the library also includes pure lines containing natural (unsubstituted) amine groups. A small amount of pure lines from each pool (eg, about a pure line (depending on the complexity of 2)) is directed against a knot-intimate sieve of a target polypeptide (or other binding target); Candidates with the same, reduced or no combination. Methods for determining binding affinity are well known in the art. 129629.doc 56- 200900079 can be used to determine the binding affinity of BIAcore surface plasma resonance analysis, which detects a binding affinity of about 2 times or more. When the starting antibody has been bound with a relatively high affinity, for example about! Λ 1 』 BIAcore is especially suitable for Kd of about 10 nM or less. In the example φ J of this paper, 4 Tianshu uses the BIAcore surface plasma resonance test. Binding affinity can be determined using Kinexa Biocensor, scintiilati〇n proximity assay ^ ELISA, (10) GEN immunoassay (IG_, fluorescence/photoreduction, camp light transfer, and/or yeast presentation. Suitable bioassays can also be used. Binding affinities for binding. In some embodiments, 'mutation-inducing methods recognized by this technique, some of which are described herein, position each amino acid in the CDR through all 20 natural amine groups. Acid substitution (one at a time in some embodiments), thereby producing a small pure line library (in each of the examples, one for each of the analyzed amine groups), each having a heterogeneity of 2 〇 members (if all 2 amino acids are substituted at each position.) Q In some embodiments, the library to be screened contains substitutions at two or more positions, which may be in the same CDR or in Two or more cDRs. Thus, a library may contain substitutions at two or more positions in one CDR. A library may be included in two or more positions of two or more CDRs Replace. Library can contain Substitutions of 3, 4, 5 or more positions found in two, three, four, five or six CDRs. Substitutions can be made using low redundancy codons. See, for example, Balint et al., (1993). ) Table 2 of Gene 137(1): 109_18). The CDR can be CDRH3 and / or CDRL3. The CDR may be one or more of CDRL1, 129629.doc-57-200900079 CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3. The CDR can be a Kabat CDR, a Chothia CDR or an extended CDR. Candidates with improved binding can be sequenced to identify CDR substitution mutants that result in improved affinity (also referred to as "modified" substitutions). Candidates for binding can also be sequenced, thereby identifying CDR substitutions that remain bound. Multiple rounds of screening are available. For example, candidates with improved binding (amino acid substitutions each comprising one or more positions of one or more CDRs) are also suitable for designing at each modified CDR position (ie, the amino acid in the CDRs) Position, where the substitution mutant displays an improved binding) a second library containing at least the original and substituted amino acids. The preparation and screening or selection of this library is discussed further below. Library scanning mutation induction also provides a means of characterizing CDRs, and the frequency of pure lines with improved binding, identical binding, reduced binding or no binding also provides information on the importance of the position of each amino acid to the stability of the antibody-antigen complex. News. For example, if the position of the CDRs remains bound when changed to all 20 amino acids, the position is identified as being less likely to be the site of antigen binding. Conversely, if the position of the CDR remains bound in only a small percentage of the substitution, the position is identified as a position that is important for CDR function. Thus, the library scan mutation inducing method produces information about the position of the CDR that can be altered to a variety of different amino acids (including all 20 amino acids) and the position of the CDR that cannot be altered or can only be changed to a minority of amino acids. Candidates with improved affinity can be combined in a second library, depending on the complexity of the desired library, including the modified amino acid at that position, the original amino acid, and can additionally include other sites at that location Substituting, or allowing the use of the desired 129629.doc -58-200900079 4 test or selection method ^ In addition, if f is desired, the position of the adjacent amino acid can be randomized to two or more amino acids. Randomization of adjacent amino acids may allow for additional conformational flexibility in the mutated CDRs, which in turn may allow or facilitate the introduction of a greater number of modified mutations. The library may also contain substitutions in the first round of screening that do not display improved affinity. Any method known in the art can be used, including the use of eight (3) "Surface Plasma Resonance Analysis Screening and the selection of any method known in the selection technique, including phage display, yeast presentation and ribosome rendering". A library member having improved and/or altered binding affinity. Detecting or selecting a second library. The invention also encompasses fusion proteins comprising antibodies (such as 9τχ and ^(7) or polymorphisms or regions or regions from the invention. In one embodiment, a fusion polypeptide comprising at least one contiguous amino acid of a variable light chain region and/or at least 10 amino acids of the variable heavy chain region is provided. In other embodiments Providing a fusion polypeptide comprising at least about 10, at least about 5, at least about 20, at least about or at least about contiguous amino acids of a variable light chain region; and/or a variable heavy chain At least about 1 , at least about 15, at least about 20, at least about 25, or at least about 3 contiguous amino acids. In another embodiment, the fusion polypeptide comprises a 9tL or 6G light chain. Variable region and/or heavy chain variable region In another embodiment, the fusion polypeptide comprises one or more CDRs of 9tl or 6G. In still other embodiments, the fusion polypeptide comprises CDR H3 and/or CDR L3 of antibody 9TL or 6G. For the purposes of the present invention The 9TL or 6G fusion protein contains one or more 9TL or 6G antibodies, respectively, and another amino acid sequence that is not attached to it in the native molecule, such as a heterologous sequence from another region of 129629.doc-59-200900079 Or homologous sequences. Exemplary heterologous sequences include, but are not limited to, tags, such as FLAG tags or 6 cents. The disclosures are well known in the art. Methods known in the art can be used. , for example, synthetically or recombinantly to produce a fusion polypeptide. Although the fusion protein of the present invention can also be used

Other methods known in the art, Sword J, Jr, C, I, for example, include chemical synthesis, but generally use the recombinant method described herein to make the fusion of the present invention by preparing a polynucleic acid encoding the same. protein. The invention also provides an antibody or polypeptide comprising and conjugated (e. g., linked) to an agent (biotin or avidin) that facilitates coupling with a solid support. For the sake of simplicity, reference will generally be made to antibodies, although it is understood that such methods are applicable to any of the Aβ binding embodiments described herein. Pick up. The suspension Us is connected to these components as described herein. The attachment can be achieved in any of a variety of ways (which is generally to immobilize the components at least for administration in a proximity association manner). For example, when t each has a substituent capable of reacting with others, a direct reaction between the agent and the antibody is possible. For example, a nucleophile such as an amine group or a sulfhydryl group may be capable of reacting with a group such as a liver or an acid group or, on the other hand, with a good leaving group (for example, a dentate group). Alkene reaction. The antibodies or polypeptides of the invention may be associated with labeling agents (also referred to as &quot;tags.&quot; such tags as such as Laughter molecules, radioactive molecules or any other label known in the art. Labeling in the art has been In general, the signal is provided (directly or indirectly). 〃 The invention also provides compositions (including pharmaceutical compositions) and kits comprising 129629.doc • 60 '200900079 antibody 9TL or 6G, and as disclosed in the present disclosure Elucidation of any of the antibodies and/or polypeptides described herein. 3 Anti-Aβ peptide with impaired effector function and polymorphism The method of the present invention uses specific binding to a β-type starch (Αρ) peptide and has a weakening effect. An antibody or polypeptide (including a pharmaceutical composition comprising the antibody or polypeptide). The other characteristics of the antibody and polypeptide are in any of the following features (- or more): (4) inhibiting the formation of a powder in the individual (b) reducing amyloid plaques in the individual's eyes; (c) treating, preventing, ameliorating or symptomatic ophthalmic diseases including, but not limited to, age-related yellow Degenerative (dry and wet), glaucoma, diabetic retinopathy (including macular edema) and other related retinal degenerative diseases; (d) significant protection or recovery of retinal function; and (e) significant visual acuity Maintained or reconstituted. The antibodies and polypeptides described herein may exhibit a desired safety profile, for example, the compositions of the present invention do not cause any significant or unacceptable levels or have any of the following: - or more : hemorrhage in the vascular structure of the brain (cerebral hemorrhage); meningoencephalitis (including altered magnetic resonance scans) increased white blood cell count in cerebrospinal fluid; central nervous system inflammation. As used herein, has &quot;weakening effect function&quot; An antibody or polypeptide that is "immunologically inert", or that is immunologically inert ("interchangeable") refers to an antibody that does not have any effector function or has reduced effector function (with antibodies that have an unmodified or naturally occurring constant region or An antibody or polypeptide of a polypeptide, for example, which is not active or has a reduced I&quot; in any one or more of the following: a) triggering complement-mediated lysis; b) stimulating antibody-dependent cell-mediated fine 129629.doc -61 - 200900079 cytotoxicity (ADCC); and c) activating micro-nerve radiance cells. Effect function activity can be reduced by about 10% Any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, and 100%. In some embodiments, the antibody and the β-class Amyloid peptide binds without triggering significant complement-dependent lysis- or target-mediated cell-mediated destruction. For example, the receptor binding site on the constant region can be modified or mutated to remove or reduce certain The binding affinity of Fc receptors such as cyRI, FcyRII and/or FcyRIII. For the sake of simplicity, reference will be made to antibodies. It should be understood that the examples also apply to polypeptides. The EU numbering system (Kabat et al. 'Sequence of Proteins of Immunological Interest; 5th edition, Public Health Service, National Institutes of Healthy, Bethesda, Md., 1991) is used to indicate which amine of the constant region (eg, of an IgG antibody) The acid residue is altered or mutated. This number can be used for a particular type of antibody (e. g., IgG(R)) or species (e.g., human). It should be understood that similar changes can be achieved across various types of antibodies and species. In some embodiments, an antibody that specifically binds to an Aβ peptide comprises a heavy chain constant region that has a debilitating effect. The heavy chain constant region can have a naturally occurring sequence or be a variant. In some embodiments, the amino acid sequence of the naturally occurring heavy chain constant region is mutated, e.g., by amino acid substitution, insertion, and/or deletion, thereby attenuating the effector function of the constant region. In some embodiments, the 1 glycosylation of the FC region of the heavy chain constant region can also be altered, e.g., completely or partially removed, thereby attenuating the effector function of the constant region. In some embodiments, the effector function is attenuated by removing the N-glycosylation of the Fc region of the anti-[beta] peptide (e.g., in the CH2 domain of IgG). In some embodiments 129629.doc-62.200900079, the N of the Fc region is removed by mutating or flanking a glycosylated amino acid residue as a residue of a glycosylation recognition sequence in the constant region. Glycosylation. Tripeptide sequence aspartic acid-X-serine (NXS), aspartic acid _ χ _ sulphonic acid (NXT) and aspartic acid χ χ _ cysteine (N_x_c) X is any amino acid other than indoleamine I) is a recognition sequence for the partial enzymatic attachment of a water-reducing compound to the aspartic acid side chain to achieve glycosylation. Mutation of any of the amino acids in the tripeptide sequence in the constant region produces glycosylated IgG. For example, σ, the N-glycosylation site N297 of human IgG1 and IgG3 can be mutated to A, D Q K or H. See Tao, 乂 M3: 2595· 2601 (1989) ‘and jeffens et al.’~ (10) Heart/Heart (4) 163:59-76 (1998). It has been reported that human igG1 and IgG3 having Gln, this or _substitution of As. 297 do not bind to human FcyR1 and do not activate complement, wherein Clq binding capacity is completely lost to IgG1 and significantly reduced for qing. In some embodiments, the amino acid n mutation in the tripeptide sequence is any of the following amino acids: A'C'D'E'F'G'Hq, i K, L, M, P, Q , R, s, T, V, W, Y. In some embodiments, the amino acid N mutation in the tripeptide sequence is a conservative substitution. In some embodiments, the amino acid X in the tripeptide sequence is mutated to proline. In some embodiments, the amino acid s is mutated to K, L, μ, N, p, Q, R, v, w, γ in the tripeptide sequence. In some embodiments, the amino acid τ in the dipeptide sequence is mutated to L, Μ, N, p, q, R, γ, w, γ. In some embodiments, the nucleotide acid c in the triad sequence is a, D, E, f, G, Η, I, K, L·, M, N, p, Q, R, V, W, γ. In some embodiments, the tripeptide is mutated to p after the amino acid of '29629.doc 200900079. In some embodiments, in an enzymatic manner (such as N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H as described in Example 3) N-glycosylation in the constant region is removed. Removal of N-glycosylation can also be achieved by the production of antibodies in cell lines with N-glycosylation deficiencies. Wright et al, J Immunol. 160(7): 3393-402 (1998). In some embodiments, the amino acid residue that interacts with the oligosaccharide attached to the N-glycosylation site of the constant region is mutated to reduce binding affinity to FcyRI. For example, F241, V264, D265 of human IgG3 can be mutated. See Lund et al., 丄 Immunology 157:4963-4969 (1996). In some embodiments, by, for example, PCT WO 99/58572 and Armour et al. 1 Molecular Immunology 40: 585-593 (2003); Reddy^, "/. i'mmwno/og'y 164:1925-1933 Modified regions as described in (2000) (such as 233-236, 297 and/or 327-331 of human IgG) to attenuate effector function. In addition to the binding domain to the target molecule, the antibodies described in PCT WO 99/58572 and Armour et al. comprise an effector domain having an amino acid sequence substantially homologous to all or part of the constant region of the human immunoglobulin heavy chain. . Such antibodies are capable of binding to a target molecule without triggering a significant complement dependent lysis, or a target cell mediated disruption. In some embodiments, the effector domain has reduced affinity for FcyRI, FcyRIIa, and FcyRIII. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or FcyRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly useful in chronic anti- 129629.doc-64-200900079 body therapy to avoid inflammatory and other adverse reactions to conventional antibody therapies. In some embodiments, the heavy chain constant region of the antibody is human heavy chain IgG1 having any one of the following mutations: 1) A327A330P331 to G327S330S331; 2) E233L234L235G236 to P233 V234A235, wherein G236 is deleted; 3) E233L234L235 to P233V234A235; 4) E233L234L235G236A327A330P331 to P233V234A235G327S330S331, wherein G236 is missing; 5) E233L234L235A327A330P331 to P233V234A235G327S330S331; and 6) N297 to A297 or any other amino acid other than N. In some embodiments, the heavy chain constant region of the antibody is human heavy chain IgG2 having the following mutations: A330P331 to S330S331. In some embodiments, the heavy chain constant region of the antibody is human heavy chain IgG4: E233F234L235G236 to P233V234A235, wherein G236 is deleted; E233F234L235 to P233V234A235; and S228L23 5 to P228E23 5 . The constant region of the antibody can also be modified to attenuate complement activation. For example, by mutating an amino acid residue in the constant region of a C1 binding motif (e.g., a Clq binding motif), complement activation of an IgG antibody can be reduced following binding of the C1 component of complement. The Ala mutation of each of D270, K322, P329, P331 of human IgG1 has been reported to significantly reduce the ability of the antibody to bind to Clq and activate complement. For murine IgG2b, the Clq binding motif constitutes residues E318, K320 and K322. Idusogie et al., /. /mw, 164:4178-4184 (2000); Duncan et al, Nature 322: 738-740 (1988). The Clq binding motifs E318, K320 129629.doc-65-200900079 and K322 identified for murine IgG2b are common for other antibody isotypes. Duncan et al.

Nature 322: 738-740 (1988). The Clq-binding activity to IgG2b can be eliminated by replacing any of the two specified residues with a residue having an inappropriate lyophilin b group in its side chain. It is not necessary to replace the ion residue with Ala to eliminate Clq binding. It is also possible to use other alkyl-substituted nonionic residues (such as Gly, lie, Leu or Val) or aromatic non-polar residues such as Phe, Tyr, Trp and Pro instead of any of the three residues. To eliminate Clq binding. Alternatively, polar nonionic residues such as Ser, Thr, Cys, and Met may be used in place of residues 320 and 322 (but not in place of 318) to eliminate Clq binding activity. The invention also provides an antibody having a weakening effect, wherein the antibody has a modified hinge region. The binding affinity of human IgG to its Fc receptor can be modulated by modification of the hinge region. Canfield et al, J. Yang, 173: 1483-1491 (1991); Hezareh et al, /. Wro/. 75: 12161-12168 (2001) ‘Redpath et al, 59: 720-727 (1998). Specific amino acid residues can be mutated or deleted. The modified hinge region may comprise a complete hinge region of an antibody derived from an antibody class or subclass different from the class or subclass of the CH1 domain. For example, the constant domain (CH1) of the IgG antibody class can be attached to the hinge region of the IgG4 antibody class. Alternatively, the new hinge region may comprise a portion of a naturally occurring chain or repeat unit, wherein the repeating units are derived from a natural chain region. In some embodiments, the natural property is altered by converting one or more cysteine residues to a neutral residue such as alanine or by converting an appropriately placed residue to a cysteine residue. Hinge area. U.S. Patent No. 5,677,425. Protein chemistry approved using this technology and 129629.doc -66- 200900079 rutting uses genetic engineering techniques and as described herein to make such changes. Polypeptides that specifically bind to the β-form and bind to the heavy chain constant region with attenuating effector function can also be used in the methods described herein. In some embodiments, the polypeptide comprises a sequence derived from an antibody 9TL or a variant thereof as shown in Table 3. In some embodiments, the polypeptide is derived from a single domain antibody that binds to an Aβ peptide. Single domain antibodies can be produced using methods known in the art. 〇midfar et al., 5ζ·〇/. 25:296-305 (2004); Herring et al., Μ 21:484 489 (2〇〇3). In some embodiments, the antibody or polypeptide is not a F(ab')2 fragment. In some embodiments the antibody or polypeptide is not a Fab fragment. In some embodiments, the antibody or polypeptide is not a single chain antibody scFv. In some embodiments, the antibody or polypeptide is a pegylated F(ab,) 2 fragment. In some embodiments, the antibody or polypeptide is a pegylated Fab fragment. In some embodiments, the antibody or polymorphism is a t-ethylated single bond antibody scFv. Other methods of making antibodies with impaired effect functions known in the art can also be used. Antibodies and polypeptides having modified constant regions can be tested in one or more assays to assess the level of effector reduction in biological activity compared to the starting antibody. For example, the ability of an antibody or polypeptide having an altered Fc region to bind to a complement or a receptor (eg, an Fc receptor on a microglial cell) or to alter the hinge region can use the assays disclosed herein and any of the The accreditation of technical approval is assessed. PCT WO 99/58572; Armour et al, Mo/ecW/ar Immunology 40: 585-593 (2003); Reddy # A &gt; J.

Immunology 164:1925-1933 (2000); Songf A &gt; Infection 129629.doc -67- 200900079 (4) 70:5 177-5184 (2002) ° In some embodiments, the antibody specifically binding to the β-class powder is more Strain antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a primatized antibody. See, for example, Yocum et al., /. Rheumatol. 25: 1257-62 (1998); Bugelski et al., //wm(10) ά5; cpehmeWa/roxz’co/oy 19:230-243 (2000). In some embodiments, the antibody is deimmunized by mutation such that the antibody does not activate the human immune system. See, for example, Nanus et al., J. Urology 170: S84-S89 (2003). As used herein, the Αβ peptide includes any fragment of an enzymatic cleavage product of a starch-like precursor protein. For example, an Αβ peptide includes any fragment of Αβ, .42 or Λβ!·43; and a peptide truncated at the terminal or c-terminus of Αβι_4(), ΑβΜ2 or Αβ!·43 via various numbers of amino acids. . The amino acid numbering used herein is based on the numbering of APmW SEQ ID NO: 17). In some embodiments, the antibody or polypeptide specifically binds to an antigen determinant within the residue of the Αβ peptide. In some embodiments, the antibody or polypeptide specifically binds to an epitope within residues 16-28 of the Ap peptide. In some embodiments, the antibody or polypeptide specifically binds to an epitope within residues 28-40 of the Αβ! peptide. In some embodiments, the antibody or polypeptide specifically binds to an epitope within residue 28 42 of the peptide. In some embodiments, the antibody or polypeptide specifically binds to an epitope within residue 28_43 of the Αβ&quot; peptide. In some embodiments, the antibody or polypeptide binds to the peptide-specific knot 129629.doc-68.200900079 and does not bind to the full-length starch-like precursor protein (APP). In some embodiments, the antibody or polypeptide specifically binds to the aggregated form of Aβ, but does not bind to the soluble form. In some embodiments, the antibody or polypeptide specifically binds to the soluble form of Aβ without binding to the aggregated form. In some embodiments, the antibody or polypeptide specifically binds to the aggregated form and soluble form of Aβ. Antibodies that bind to various aggregate forms of Aβ, such as antibodies that bind to a starch-like β-derived diffusible ligand (ADDL), and antibodies that bind to starch-like fibrils and/or deposits, are known in the art. WO 03/104437; U.S. Publication No. 2003/0147887; U.S. Publication No. 2004/0219146. In some embodiments, the antibody or polypeptide comprises one, two or three CDRs from the 3D6 immunoglobulin light chain (SEQ ID NO: 2 in US Publication No. 2003/0165496 or 2004/0087777) and/ Or one, two or three CDRs from a 3D6 immunoglobulin heavy chain (SEQ ID NO: 4 in US Publication No. 2003/0165496 or 2004/0087777). In some embodiments, the antibody or polypeptide comprises a variable heavy chain region as set forth in SEQ ID NO: 8 of US Publication No. 2003/0 165496 and SEQ ID: 5 in US Publication No. 2003/0165496 The variable light chain region is set forth in the section. In some embodiments, the antibody or polypeptide comprises a variable heavy chain region as set forth in SEQ ID NO: 12 of US Publication No. 2003/0165496 and in SEQ ID NO: 11 of US Publication No. 2003/0165496 Clarify the variable light keypad. In some embodiments, the antibody or polymorphism comprises one, two or three 129,629 from the 10D5 immunoglobulin light chain (SEQ ID NO: 14 in US Publication No. 2003/0165496 or 2004/0087777) .doc -69- 200900079 CDRs and/or one, two or three CDRs from a 10D5 immunoglobulin heavy chain (SEQ ID NO: 16 in US Publication No. 2003/0165496 or 2004/0087777). In some embodiments, the antibody or polypeptide specifically binds to an epitope within residues 33-40 of Αβ,^. In some embodiments, the antibody or polypeptide specifically binds to an epitope on the Αβ^ of amino acid 35-40. In some embodiments, the antibody or polypeptide specifically binds to an epitope comprising the amino acid 36-40. In some embodiments, the antibody or polypeptide specifically binds to an epitope comprising an amino acid 39 and/or 40. In some embodiments, the antibody or polypeptide specifically binds to Aβ, .40, but does not specifically bind to Aβ 1-42 and/or Aβ.43. In some embodiments, the antibody or polypeptide is an antibody 9TL as described herein or an antibody or polypeptide derived from a 9TL. In some embodiments, the antibody or polypeptide competitively inhibits binding of the antibody 9TL and/or the antibody or polypeptide derived from 9TL to Αβ. In some embodiments, the antibody is not an antibody 22 86 as described in PCT WO 2004/032868. In other embodiments, the antibody or polypeptide is an antibody 6G as described herein or an antibody or polypeptide derived from 6G. In some embodiments, the antibody or polypeptide competitively inhibits binding of antibody 6G and/or antibody or polypeptide derived from 6G to Αβ!, and AP _42. In some embodiments, the antibody is not the antibody 2294 described in US 2004/0146512 and WO 04/032868. Antibody 2294 binds to an antigenic determinant very similar to antibody 6G as described in WO 20061 18959. Methods for making antibodies and polypeptides are known in the art and described herein. 0 129629.doc -70- 200900079 ... 1 The same or spatially overlapping epitopes can be used for competition testing Whether two antibodies bind to the same epitope, or an antibody combines another antibody with an antigen. These checks are known in the art. The antigen is typically immobilized on a multiwell plate and the ability of the unlabeled antibody to block the binding of the 払5-hex antibody is measured. A common marker for such competition assays is radioactive or enzymatic labeling. Polynucleic Acid, Vectors, and Host Cells The present invention also provides isolated polynuclears encoding the antibodies and polypeptides of the invention (including antibodies comprising the polypeptide sequences of the light and heavy chain variable regions of Figures i and 8) Bittern' and the vector and host cell comprising the polynucleotide. Thus the invention provides a polynucleotide (or composition, including a pharmaceutical composition) comprising a polynucleotide encoding any of the following: (hook antibody 9TL or 6G or Table 3 and Table 8) a variant thereof; (b) a fragment or region of the antibody 9TL or 6G or a variant thereof shown in Table 3 and Table 8; (c) antibody 9TL or 6G or the one shown in Table 3 and Table 8. The light chain of the variant; (d) the heavy chain of the variant 9TL or 6G or the variants shown in Table 3 and Table 8; (e) the variation from the antibody 9TL or 6G or the changes shown in Table 3 and Table 8. One or more variable regions of the light chain and/or heavy chain of the body; (f) one or more CDRs (-, two, three) of the antibody 9TL or 6G or the variants thereof shown in Tables 3 and 8. , four, five or six CDRs; (g) CDR H3 from the heavy chain of antibody 9TL or 6G; (h) light chain CDRs from antibodies 9TL or 6G or variants thereof shown in Tables 3 and 8. L3; (1) Three CDRs from the light chain of the antibody 9TL or 6G or its variants shown in Table 3 and Table 8; (j) Variants from antibody 9TL or 6G or Table 3 and Table 8 Three CDRs of the heavy chain; (k) from antibody 9TL or 6G or Table 3 and 129629.doc - 71 - 200900079 The three CDRs of the light chain of the variants in Table 8 and the three CE) r of the heavy chain from the antibody 9TL or 6G or the variants shown in Table 3 and Table 8; and (1) An antibody of any of the groups 3 (b) to (k). In some embodiments, the polynucleotide comprises any one or both of the polynucleotides set forth in SEQ ID N:9, SEQ ID NO:10, SEQ ID NO:34, and SEQ ID NO:35 By. In another aspect, the invention provides a polynucleotide encoding any of the antibodies (including antibody fragments) and polypeptides (such as antibodies and polypeptides having attenuating effector function) as described herein. Polynucleic acid can be obtained by procedures known in the art. In another aspect, the invention provides a composition (such as a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises a performance vector comprising a polynucleotide encoding a 9TL antibody as described herein. In other embodiments, the compositions comprise a performance vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In still other embodiments, the composition comprises either or both of the polynucleotides shown in seq ID NO: 9 and SEQ ID N: l. The expression vectors, as well as the administration of the polynucleotide compositions, are further described herein. In another aspect, the invention provides a method of making any of the polynucleotides described herein. Polynucleotides complementary to any such sequences are also contemplated by the invention. Polynucleosides can be single-stranded (coding or antisense) or double-stranded and can be DNA (genomic DNA, CDNA or synthetic biliary) or RNA molecules. The RNA molecule includes an HnRNA molecule containing an intron and corresponding to the DNA molecule in a one-to-one manner, and an mRNA molecule containing no intron of 129629.doc-72-200900079. Other coding or non-coding sequences may, but need not, be present in the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or branch materials. A polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or a variant that may comprise such a sequence. The polynucleotide variant contains one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect of immunoreactivity on the encoded polypeptide can generally be assessed as described herein. Preferably, the agonist exhibits at least about 70% homogeneity, more preferably at least about 8% consistency, and most preferably at least about 90% homogeneity to the polynucleoside oxime sequence encoding the native antibody or portion thereof. Two nucleotide or polypeptide sequences are referred to as 'consistent' if the nucleotide or amino acid sequence in the two sequences is identical when aligned for maximum correspondence as described below. Comparing sequences on a comparison window to identify and compare local regions of sequence similarity, thereby performing a comparison between the two sequences. As used herein, "comparison window" refers to a segment of at least about 2 adjacent positions (usually 30 to about 75, 4 〇 to about 5 (), after the two sequences are optimally aligned, the sequence can be compared to a reference sequence having the same number of contiguous positions. The best sequence for comparison Alignment can be performed using the MegaUgn program in the suite of bioinformatics software (DNASTAR, lnc·, Madison, surgery) using preset parameters. This program reflects several alignment schemes described in the following references; Dayh〇 Ff,Μ〇. Ο”” A model of evolutionary change in proteins-Matrices f〇r 129629.doc -73· 200900079 detecting distant relationships. In Dayhoff, Μ.0. (ed.) Atlas of Protein Sequence and Structure, N Ational Biomedical Research Foundation, Washington DC Vol. 5 &gt; Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Volume 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971 , Comb. Theor. 11:105 ; Santou, N., Nes, M., 1987,

Mol. Biol, Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA;

Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730. Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences on a comparison window having at least 20 positions, wherein the two sequences are optimally aligned with the reference sequence (which is not The portion comprising the addition or deletion of the polynucleotide or polypeptide sequence in Comparative View® may comprise 2% or less 'through ten 5°/〇 to 15°/. Or 1 〇·&gt;/〇 to 12. /. Add or delete (ie, missing). The percentage is calculated by determining the number of positions in the same sequence of the same nucleic acid base or amino acid residue in both sequences to produce the number of matching positions divided by the number of matching positions by the total number of positions in the reference sequence (ie, meat Size) and multiply the result by 1〇〇 to produce a percent sequence identity. The variant may also (or) be substantially the same as the native gene or part thereof or complement. 129629.doc •74· 200900079 Source. Such polynucleotide variants are capable of hybridizing to naturally occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.

Suitable "moderately stringent conditions" include pre-washing in a solution of 5xSSC, 0.5% SDS, u mM EDTA (pH 8.0); overnight at 5 (rc_65〇c, 5xSSCT hybridization; followed by two washes at 65 °C) 2 〇 minutes, 2χ, 〇.5χ and 0.2xSSC (containing 0.1% SDS). As used in this article, &quot;highly stringent conditions&quot; or &quot;high stringency conditions&quot; are as follows, (1) Washing with low ionic strength and high temperature, for example, using 0.015 Μ sodium chloride / 0.0015 Μ sodium citrate / 〇 · 1% sodium lauryl sulfate at 5 (rc); (2) using a denaturing agent during hybridization, such as Indoleamine, for example at 42. underarm with 50/〇 (ν/ν) metformin and 01% bovine serum albumin/〇1〇/. Sucrose (Ficoliyo.l% polyvinylpyrrolidone/5〇mM Sodium phosphate buffer 2η 6.5), and 750 mM sodium sulphate, 75 mM sodium citrate, or (3) 50% methotrexate, 5xSSC (〇75 M Naa, 〇〇75 PCT citric acid at 42 C) Sodium), 50 mM sodium phosphate (pH 6_8), 〇1% sodium pyrophosphate, 5 χ丹哈伊德 solution (Denhardt, ss〇luti〇n), ultrasonically treated squid sperm DNA (50 pg/mi), 〇1% S DS and 1% dextran sulfate, washed with 2xSSC (sodium chloride/sodium citrate) and washed with formazan at 55 滁, followed by EDtA at 55 Between the lxSsc composition is strictly washed. Those skilled in the art will recognize how to adjust the temperature, ionic strength, etc. necessary to accommodate the factors of the length of the scorpion and its similar factors. The slave technician should understand the genetic code. Simple, therefore there are a variety of genomic DNA sequences as described herein. Some of these polynuclears 129629.doc -75- 200900079 glycosides with any scorpion, ',,, soil The minimal homology of the nucleotide sequence. However, this day-to-day transformation covers the gene of the polynucleic acid sequence provided herein by the difference in codon usage. The gene is within the scope of the invention. The allele is an internal (four) factor and is altered by one or more mutations, such as deletions, additions and/or substitutions of nuclear phytic acid. The resulting mRNA and protein may (but not Required) to have a changed structure or work. Standard techniques can be used ( Alleles are identified, such as by hybridization, amplification, and/or library sequence comparison. The polynucleotide of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis is well known in the art. The methods are not necessarily described in detail herein. Those skilled in the art can use the sequences and commercial DNA synthesizers provided herein to produce the desired DNA sequences. RNA is obtained by isolating the DNA in a suitable vector and inserting it into a suitable host cell. When the cell replica and DNA are transcribed in the % of RNA, they can then be isolated using methods well known to those skilled in the art, for example, as set forth by Sambrook et al. (1989). i Suitable selection vectors can be constructed according to standard techniques or can be selected from a wide variety of selection vectors available in the art. The expression vector is typically a replicable polynucleotide construct comprising a polynucleotide according to the invention. It indicates that the expression vector must be replicable in the host cell as a discrete portion of the chromosomal DNA or chromosomal DNA. Suitable expression vectors include, but are not limited to, plastids, viral vectors including adenovirus, adenovirus, reversal virus, viscous body, and expression vectors disclosed in PCT Publication No. WO 87/04462. 129629.doc -76- 200900079 by any of a number of suitable Fang Tian Wan, including electroporation, calcium, barium chloride, phosphonium & π wiped with lanthanum strontium calcium, strontium-glucan or other Substance · Microprojectile bombardment; lipid transfer?转之转木, 转木 (hpofectmn); and infection (such as vaccinia table + a * 』 such as infectious agents with intermediate poisoning), can be introduced into the host cell containing the relevant vector. The nucleus is also provided with host cells comprising any of the polynuclei (IV) described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CH〇 cells. See also PC Ding Publication No. 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli® or Bacillus subtilis) and yeast (such as Saccharomyces cerevisiae (4) W), Schizosaccharomyces pombe (10) (IV); or Kluyveromyces cerevisiae ([/ (10) The host cell preferably expresses the eDNA at a level that is about 5 times, more preferably (7) times, or even more preferably 2 G times higher than the level of the endogenous antibody or protein (if present) of the corresponding endogenous cell of interest in the host cell. Immunoassay or FACS to achieve screening of host cells that specifically bind to Αβ, can overexpress cells of the antibody or protein of interest. Diagnostic use of 9TL or 6G-derived antibodies and anti-Aβ antibodies with impaired effector function The antibody 9TL or 6〇 binding to the C-terminus of one or more Aβ peptides can be used to identify or detect the presence or absence of the target Aβ in the eye. For simplicity, it will usually be referred to for 9TL or 0G antibodies, but It is understood that these methods are applicable to any of the Αβ binding examples (such as polypeptides) described herein. Detection 129629.doc • 77- 200900079 generally includes binding biological samples to Αβΐ 4() The antibody described herein is contacted, and forms a complex with 特异性β, which specifically binds to an antibody of VIII (eg, 9tl). The formation of this complex may be in vitro or in vivo. As used herein, the term is used herein. &quot;Detection•• Includes 疋 and/or quantitative 彳贞 (measurement level) with or without reference photographic. Any of a variety of known methods can be used for detection, including ( But not limited to, an immunoassay that uses an antibody that binds to a polypeptide, such as by an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and the like; and a functional assay for the encoded polypeptide, For example, binding activity or enzymatic assays. The anti-system is detectably labeled when a 丨φ &gt; μ-beta-col τ is also implemented. Other embodiments are known in the art and described herein. The antibodies and polypeptides can be used to (4) diagnose, and monitor an ophthalmic disease, condition, or condition having altered or abnormal Αβ or βΑΡΡ expression. Thus, in some embodiments, the present invention provides a method comprising causing a change in the eye to be suspected A sample (sample) of an individual with abnormal Αβ expression is contacted with an antibody or polypeptide of the present invention, and whether the level of the (tetra) peptide is different from the Αβ peptide level of the control or comparative sample. In other embodiments, the present invention provides a method Contains samples (samples) that are in contact with individuals and measures AJ3 performance levels. For diagnostic applications, antibodies can detect partial markers including, but not limited to, radioisotopes, fluorescent labels, and various enzymes. Qualitative labeling. Methods for ligating a label to an antibody are known in the art. In other embodiments of the invention, the antibody of the invention does not require labeling and the pot is present in a labeled antibody that can bind to the antibody of the invention. The antibody of the present invention can be used in any of the six known test tissues, such as the competitive office 129629.doc-78-200900079 combined test, direct and indirect sandwich test and immune sedation test. Z〇ja,

Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). Antibodies can also be used for in vivo diagnostic assays, such as in vivo imaging. The antibody is typically labeled with a radionuclide (such as (1) In, &quot;Tc, &quot;C,, (2) ut3H) so that the cell or tissue of interest can be localized using immunoscintigraphy. Antibodies can also be used as stains in pathology according to techniques well known in the art. Anti-Aβ antibodies with impaired effect function can be used to measure retinal function to diagnose individuals at risk of retinal-related degenerative ophthalmic diseases or diagnosed with retinal-related degenerative ophthalmic diseases, and to assess the progression of any treatment and disease. In some embodiments, an anti-Aβ antibody having a weakening effect is administered to an individual and the level of Aβ in the plasma is measured, whereby an increase in plasma Aβ indicates the presence of a brain-like starch load in the individual and/or Or level. These methods can be used to monitor the effectiveness of the treatment and the stage of the disease and to determine future administration and frequency. Antibodies with attenuating effect function may have a better safety profile and provide advantages for such diagnostic uses. Methods of Using Anti-Aβ Antibodies for Therapeutic Use The antibodies (including polypeptides), polynucleotides, and pharmaceutical compositions described herein are useful in methods of treating, preventing, and inhibiting the development of ophthalmic diseases characterized by retinal-related degeneration. Such methods comprise administering to the individual an effective amount of an antibody that specifically binds to a protein or protein deposit or a polynucleotide encoding the antibody, wherein the antibody has an attenuating effector function. The antibodies (including polypeptides), polynucleotides, and pharmaceutical compositions described herein can be used in a method for treating, preventing, and inhibiting the progression of age, the scallop &amp; Diseased (wet and dry),: light; his eye diseases such as age-related macular degeneration macular edema, jp diabetic retinopathy (including diabetes r η plaque edema), cloth t, ##,,,〇 Gg Duns myopia degeneration, ocular tumors and pita-related retinal degenerative diseases, such as Μ-body, multi-M i ^, etc., involve administering to the individual an anti-Xi peptide or a polynucleotide or a gluteal drug 1 mm. In the case of preventive response, it is sufficient to divide or reduce the risk of salt and phlegm and to reduce the severity of the disease or delay the onset of the disease.

Injecting a pharmaceutical composition or drug into a patient who is susceptible to an ophthalmic disease or otherwise at risk of an ophthalmic disease, including the biochemical, tissue, and/or behavioral symptoms of the disease The complication and intermediate pathology presented during development are applied to the suspected or already suffering from an amount sufficient to cure or at least partially arrest the symptoms of the disease (biochemical, tissue and/or behavioral symptoms). Patients of this disease are administered compositions or drugs that include complications and intermediate pathological phenotypes in the development of the disease. V. Complications of age-related macular degeneration and intermediate pathological phenotypes including thick diffuse visual network subcutaneous pigment epithelial (RPE) deposition, lip-rich drusen-like deposit, Bruch Membrane thickening, spotting of RPE atrophy, and choroidal neovascularization. The invention also provides a method of delaying the development of retinal degeneration and/or its symptoms in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide described herein. The invention also provides a method of restoring or protecting the function of the retina of an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide described herein. 129629.doc -80- 200900079 The invention also provides a method of reducing amyloid plaques and/or reducing or slowing Αβ accumulation in an individual retina comprising administering to the individual an effective amount comprising an antibody, polypeptide or poly A pharmaceutical composition of nucleotides. The invention also provides a method of removing or eliminating amyloid plaque and/or Αβ accumulation in an individual's retina comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide described herein. In some embodiments, the amyloid plaque is in the brain of an individual. The present invention also provides a method for reducing 积累β peptide accumulation in Α3 peptide, inhibition and/or eclipse retinal tissue in a reticular tissue, comprising administering to an individual an effective antibody comprising the antibody or polypeptide described herein. Or a pharmaceutical composition of a polynucleotide. The Aβ polypeptide may be in a soluble, oligomeric or deposited form. The oligomeric form (9) comprises 2-5 Α Αβ polypeptides which may be a mixture of full lengths and squads and/or any truncated version of such peptides. The present invention also provides a method for improving or reversing the retinal degeneration in an ophthalmic disease.

The invention comprises administering to a subject an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleic acid as described herein. The invention also provides a method of treating or preventing a disease associated with deterioration of the retina, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an aggregation with a beta-beta peptide or a beta-class peptide peptide Form-specific binding

The antibody, wherein the antibody comprises an FC region having a variation with a naturally occurring Fc region, wherein the mutation causes a weakening effect function whereby the administration of the antibody produces microbleeds that are smaller than the administration of the non-mutant antibody. By means of a single point at a single time point or multiple time points, .....-T true shape/worker first one or more parts, this method can be realized (including prevention methods or treatment 129629) .doc -81 - 200900079 Law). . It can also be administered to multiple sites almost simultaneously. The frequency of administration can be in the therapy "Wang::: confirm and adjust, and the frequency of administration is based on achieving the desired results. In seven cases, continuous continuous release of antibodies (including peptides), polynucleotides and their ligands The pharmaceutical compositions of the present invention may be suitable. Various formulations and devices for achieving sustained release are known in the art. Patients, individuals or individuals include mammals 1 canine, pig and sheep animals. It may be human or may be afflicted or not ($ suffering from disease or symptoms shown herein. The method of the present invention may be administered prophylactically - without further risk assessment for individual patients. The method of the present invention is suitable for breaking An individual with known genetic risk of age-related macular degeneration. Such individuals include individuals with relatives who have experienced the disease and individuals who have determined the risk by analyzing genetic or biochemical markers. Pharmaceutical compositions useful in the above methods Included in any of the antibodies, polymorphisms and/or polynucleotides described herein. In some embodiments, the antibody is an antibody 9TL or 6G or a variant thereof as shown in Tables 3 and 8. In some embodiments, the antibody is an antibody that specifically binds to the Aβ peptide and comprises a constant region having a weakening effect. The administration and dosage of the antibody are preferably administered to the mammal in a carrier, preferably a pharmaceutically acceptable carrier. Dosing. Suitable carriers and their formulations are described in: and (iv) (10) 4 corpses/mr, similar, 18th edition, a Gennar, ed., "Publishing Co., Easton, pa, 1990; and mail called iSWewce, 20th edition, Mack

Publishing, 2000. A suitable amount of a pharmaceutically acceptable salt is usually employed in the formulation 129629.doc -82 - 200900079 to render the formulation isotonic. Examples of the carrier include saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8' and more preferably from about 7 to about 7.5. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing antibodies, and matrices such as shai are in the form of shaped articles&apos; such as films, liposomes or microparticles. Those skilled in the art will appreciate that certain carriers may be preferred depending on, for example, the route of administration and the concentration of antibody administered. By injection (for example, systemic, intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal, intracerebral, intraventricular (intracerebralVentricular) and intranasal) or by other methods (such as rounds) It is delivered to the bloodstream in an effective form, and antibodies can be administered to mammals. Antibodies can also be administered by isolating perfusion techniques, such as isolating tissue perfusion, to exert a local therapeutic effect. Further, the antibody of the present invention can be administered to the eye locally or in the form of an injection such as intravitreal injection, subretinal injection or double-sided injection. Additional information on administering compounds to the eye can be found in T〇lentin〇 et al., et al. 24 (2 132 132-138, Reich et al., ed/ecw/ar νζϋ/ί 9 (2003) 210-216 Lieutenant may determine the effective dose and schedule of administration of antibodies by experience, and such decisions are within the skill of the art. Those skilled in the art should be aware that the dose of antibody that must be administered will be considered (for example) The antibody used in mammals, the antibodies used in the 疋k彳二疋 type, and other drugs administered to mammals. Guidance for selecting the appropriate dose for antibodies can be found in the literature on antibody therapeutic _, such as Handb 〇〇k 〇f m(10)丨(10)^ 129629.doc -83- 200900079 Do οί/ies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ, 1985, Chapter 22 and pages 303-357; Smith et al. Antibodies in Human Diagnosis and Therapy, Yidibeic, ed., Raven Press, New York, 1977, pp. 365-389. Depending on the factors mentioned above, the typical daily dose range for antibodies used alone can range from 1 per day. Pg/kg up to 1 〇mg/kg body weight or more. In general, any of the following dosages can be used: administration of at least about 5 mg/kg body weight; at least about 10 mg/kg body weight; at least about 3 mg/kg body weight; At least about ! mg/kg body weight; at least about 750 pg/kg body weight; at least about 500 pg/kg body weight; at least about 250 pg/kg body weight; at least about 1 〇〇 gg/kg body weight; at least about 50 pg/kg body weight; At least about 1 〇/kg body weight; at least about i called / "weight or more dose. At the beginning of treatment" antibodies can be administered at lower doses or less frequently to avoid potential side effects, such as temporary brain starch vessels Lesions (CAA). In some embodiments, more than one antibody may be present. Such compositions may contain at least one, at least two, at least three, at least four, at least five different antibodies (including polypeptides) of the invention. The antibody can also be administered to a mammal in combination with an effective amount of one or more other therapeutic agents. The antibody can be administered sequentially or simultaneously with the or other therapeutic agent. The amount of antibody and therapeutic agent depends, for example, on the type of use. Drug, pathological condition treated and Depending on the schedule and route of administration, the amount will generally be small if each is used individually. After administration of the antibody to a mammal, the physiological conditions of the mammal can be monitored in a variety of ways well known to those skilled in the art. 129629.doc -84- 200900079 The above principles and dosages can be adapted to the polypeptides described herein. Polynucleotides encoding the antibodies or polypeptides described herein can also be used to deliver and express antibodies or polypeptides in a desired cell. Obviously, expression vectors can be used to directly express antibodies. The expression vector can be administered systemically, intraperitoneally, intravenously, intramuscularly, subcutaneously intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, transdermally or by inhalation. For example, expression carrier administration includes topical or systemic administration, including injection, oral, granular or insertion catheter administration, and topical administration. Those skilled in the art are familiar with the administration of performance carriers to obtain in vivo expression of exogenous proteins. See, for example, U.S. Patent No. 6,436,908; 6,413,942; and 6,376,471. Targeted delivery of a therapeutic composition comprising a polynucleotide encoding an antibody of the invention can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al. '7&gt; (9) Heart (1993) 1 1:202; Chiou's 'Gene Therapeutics: Methods And Applications Of Areci 7&gt; (10) (edited by ja Wolff) (1994) Wu et al., price 〇/· CTzem. (1988) 263:621; Wu et al., 乂仏〇/. C/z Hidden (1994) 269:542; Zenke et al., TVai/· dcac/. &lt;;SW. (1990) 87.3655, Wu et al., /. _g/o/· C/zem. (1991) 266:338. In a gene therapy regimen, a therapeutic composition containing a polynucleotide is administered in a range of from about 100 ng to about 2 mg of DNA for topical administration. A concentration range of from about 5 ng to about 5 〇 mg, from about 盹 to about 2 mg, from about 5 pg to about 5 Å, and from about 2 至 to about 1 〇〇 gg DNA may also be used during the gene therapy regimen. Therapeutic polynucleosides of the invention can be delivered using a gene delivery vehicle 129629.doc •85- 200900079 Acids and polypeptides. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Ccsmcer Ge (1994) 1:51; Kimura,

Human Gene Therapy (1994) 5:845 » Connelly, Human Gene (1995) 1:185; and Kaplitt, iVaiwre Genei/cs (1994) 6:148). Expression of such coding sequences can be induced using an endogenous mammalian promoter or a heterologous promoter. The expression of the coding sequence can be constitutive or modulated. That is, a viral-based vector for transmitting the desired polynucleotide and expressing it in a desired cell is well known in the art. Exemplary viral vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication No. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/1 1230; WO 93/10218; WO 91/02805; US Patent No. 5,219,740; No. 4,777,127; British Patent No. 2,200,651; and EP 0 345 242), alpha-viral vector ( For example, Sindbis virus vector, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) And Venezuela equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adenovirus (AAV) vectors (see, for example, PCT disclosure) WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/1 1984 and WO 95/00655. The drug 129629.doc-86-200900079, which is linked to the killing adenovirus, as described in Curiel, //wm, 77?er. (1992) 3:147, may also be used. Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polyvalent cation condensation DNAs linked to or not linked to the adenovirus alone (see, for example, Curiel, //Wm, Ge-like 77^γ. 1992) 3:147); ligands link DNA (see, for example, wu, "/. Xenopus./. (1989) 264:16985);

Eukaryotic cell delivery vehicle cells (see, for example, U.S. Patent No. 5,814,482; PCT Publication No. WO 95/97072; WO 96/17072; WO 95/3,763 and WO 97/42338) And nuclear charge neutralization or fusion with cell membrane. Naked DNA can also be used. An exemplary naked dna introduction method is described in PCT Publication No. WO 90/1 1092 and U.S. Patent No. 5,58,859. Liposomes that act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication No. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968; . in

Other methods are described in Philip, Mo/. Ce//, (1994) 14:241 1 and in Woffendin, w. (1994) 91:1581. Kits The invention also provides articles and kits comprising the substances described herein for use in the treatment of ophthalmic diseases, such as age-related macular degeneration (wet and dry), glaucoma, diabetic retinopathy (including diabetes) Macular edema, Bruch's membrane rupture, myopia degeneration, eye tumors and other related retinal degenerative diseases. The article comprises a container having a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from a variety of materials such as glass plastic. The container holds a composition with an active agent that is effective for treating a pathological ophthalmic condition or for detecting or purifying 129629.doc -87.200900079. The active agent in the composition is an antibody and preferably comprises a monoclonal antibody specific for Aβ or βΑΡΡ. In some embodiments, the active agent comprises antibody 9TL or 6G or any antibody or polypeptide derived therefrom. In some embodiments, the 'active agent' comprises an anti-Aβ antibody or a polymorphism having a weakening effector function. In some embodiments, the anti-Aβ antibody or polypeptide comprises a heavy chain constant region, wherein the constant region has attenuating effector function. The label on the container indicates that the composition is for the treatment of pathological ophthalmic conditions such as AMD and may also indicate guidance for in vivo or in vitro use, such as those described above. The invention also provides kits comprising any of the antibodies (such as 91^ or 6g), polypeptides, and polynucleic acids described herein. In some embodiments, the kit of the invention comprises the container described above. In other embodiments, the kit of the present invention IS: a second container containing a buffer. It may additionally include other substances required by the user's point of view, including its Mg diluent, over, production crying, including other buffers, and two, needles, syringes, and packaging inserts, such packaging. Insert i is used in any of the methods described in the text, such as the method for treating gamma to inhibit or reduce the accumulation of brain Αβ peptide, for the detection or purification of Αβ or β ΑΡΡ in the kit, antibody wanted四 (4) markers such as radioisotopes, fluorescent species, _ mixture, bioluminescent luminescent composition (here: Ming 2: Ben: any of the methods described in the manufacture. '', The following examples are provided to illustrate, but not limit, the invention. Poor I29629.doc • 88- 200900079 Example 1. Binding affinity determination of antibody 9TL and its variant A. General method A general method was used in this example. Fab fragments for expression vector expression for clone characterization are similar to Barbas (2001) P/zage display: a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press pg 2.10. Vec Under the control of the IPTG-inducible lacZ promoter of the promoter in tor pComb3X), however, the modification includes the addition and expression of the following other domains: human kappa light chain constant domain of IgG2a human immunoglobulin and CHI constant domain, Ig γ-2 chain Area C, protein registration number 859 01859; immunoglobulin κ light chain (homosapiens), protein accession number CAA09181. Small scale Fab preparation Fab small scale performance in 96 well plates was performed as follows. Starting with E. coli transformed from the Fab library, colonies were picked to inoculate the mother plate (agar LB + Ampicillin (50 pg/ml) + 2% glucose) and the working plate (2 ml/well, 96 wells/ Plate containing 1.5 mL LB + ampicillin (50 pg/ml) + 2% glucose). Both plates were grown at 30 ° C for 8-12 hours. The mother plates were stored at 4 ° C and the cells from the working plates were granulated at 5000 rpm and resuspended in 1 mL LB + ampicillin (5 〇pg/ml) + 1 mM IPTG to induce Fab expression. . The cells were harvested by centrifugation after 5 h of performance time at 30 ° C, and then resuspended in 500 pL of buffer HBS-P (10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 0.005°). /〇P20). The lysis of HBS-P resuspension fine 129629.doc -89- 200900079 was achieved by cold; East (-80 C) followed by a cycle of melting at 37 °C. The cell lysate was centrifuged at 5000 rpm for 3 〇 min to separate cell debris from the supernatant containing Fab. The supernatant was then injected into a BIAc〇re plasma resonance device to obtain affinity information for each Fab. The Fab was rescued from the mother plate to sequence the DNA and was used for large-scale pab fabrication and detailed characterization as described below. Large scale Fab preparation To obtain detailed kinetic parameters, the Fab was expressed and purified from large cultures. A 5 mL overnight culture from the selected Fab-expressing E. coli strain was used to inoculate an Erlenmeyer flask containing 200 mL of LB + ampicillin (5 〇 pg/ml) + 2% glucose. The pure line was incubated at 30 °C until the OD55Gnm of i.o was reached, and then induced by replacing the medium with 200 ml of LB + ampicillin (50 gg/ml) + 1 mM IPTG. After 3 (rCT5 h performance time, the cells were granulated by centrifugation, and then resuspended in 1 mL of PBS (pH 8;) by freezing/thawing (in _80. (: and 37, respectively) The lysate of the cells was obtained in two cycles of (:). The cell lysate was loaded on a solution of Ni-NTA super-lipose (Qiagen, Valencia. CA) equilibrated with pBS (pIi 8). The column was then washed with 5 column volumes of PBS (pH 8). Individual Fabs were dissolved in different dissolving fractions with pBS (pH 8) + 3 mM mM imidazole. Dissolution containing Fab was pooled in PBS. Dialysis, followed by ELISA quantification' followed by affinity characterization. Fully antibody preparation to express whole antibodies, heavy and light chain variable regions were selected in mammalian expression vectors and lipid transfected amines were used ( Lip〇fectamine) was transfected into HEK 293 cells for transient expression. The antibody was purified using the standard method using protein 129629.doc -90-200900079 A. Vector pDb.9TL.hFc2a is a heavy chain containing 9TL antibody The vector is suitable for the transient or stable performance of the heavy chain. The vector pDb.9TL.hFc2a has the corresponding region Nucleotide sequence: murine cell giant virus promoter region (nucleotide 丨_612); synthetic intron (nucleotide 619-1 507); DHFR coding region (nucleotide 707-1067); human Growth hormone signal peptide (nucleotide 1 525-1 602); heavy chain variable region of 9TL (nuclear bud acid 1603-1951) 'Human heavy chain igG2a constant region containing the following mutations: Α33〇Ρ331 to S33〇S331( Refer to the amino acid number of the wild-type lgG2a sequence; see Eur. J_ Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal (nucleotide 2960-3203); SV40 enhancer region (nucleoside Acid 3204-3449); phage fl region (nucleotide 3537_4992) and beta indolease (AmpR) coding region (nucleotide 4429_5286). The vector pDb_9TL.hFc2a was stored in ATCC on July 2nd, 20th And designated as ATCC accession number PTA-6124. The vector pEb.9TL.hK is a expression vector containing the light chain of the 9TL antibody and is suitable for transient expression of the light chain. The vector pEb.9TL.hK has nucleotides corresponding to the following regions Sequence: murine cell giant virus promoter region (nucleotide 612); human EF_1 intron (nucleotides 611-1142); human growth hormone signal (nucleotides 1173-1150); antibody 9TL light chain variable region (nuclear acid 125 1-1593); human 1 &lt; chain constant region (nucleotides 1594-1914); SV40 late polyadenylation signal (nucleus Glycosidic acid 1932-2175); SV40 enhancer region (nucleotides 2176-242 1); phage η region (nucleotides 2509-2964) and β-prolylase (AmpR) coding region (nucleotides 3401-4258 ). The vector pEb.9TL.hK was saved at the ATCC on July 20, 2004 and designated as ATCC accession number pTA_129629.doc • 91 · 200900079 6125.

Biacore assay The affinity of the 9TL monoclonal antibody was determined using the BlAcore 30(R)TM Surface Plasma Resonance (SpR) system (BIAc〇re, INC, Piscaway NJ). One method of determining affinity is to immobilize 9TL on (3M5 wafer and measure Αβ), the binding kinetics of the peptide to the antibody. According to the supplier's instructions, Ν_ethyl_Ν, _(3_ diamidopropyl CM5-carbodiimide hydrochloride (EDC) &amp; N-hydroxysuccinimide (NHS) activates CM5 wafers. Antibody 9TL or variants thereof are diluted in 10 mM sodium acetate (pH 4.0 or 5.0) and Injections were made on the activated wafer at a concentration of 0.005 mg/mL. Using variable flow times through individual wafer channels, antibody densities were achieved in the following ranges: 丨〇〇〇_2〇〇〇 reaction units (Ru) or 2000_3〇〇 〇Reaction unit (Ru). Block the wafer with ethanolamine. The regeneration study showed that the solution containing 2 volumes of PIERCE Dissolution Buffer and i volume of 4 M NaC1 effectively removed the bound AP1_4() peptide while maintaining the activity of 9TL on the wafer.

Adhere to more than 200 injections. HBs_Ep buffer (〇〇1 M HEpEs (pH 7.4) '0.15 M NaCU' 3 mM EDTA, 0.005% surfactant P20) was used as an electrophoresis buffer for all BIAcore assays. The purified Αβι, a serial dilution of the synthetic peptide sample (01_1〇χ estimated 〇) was injected at 1〇〇^L/min] min and allowed to dissociate for 1 〇 min. By fitting the data to ι:ι

Langmuir combined model (Karlss〇n, R R〇〇s, H. (four) (10) shirt, L

Petersson, B. (1994). Methods Enzymology 6. 99-110) Using the BIAevaluation program to simultaneously obtain the kinetic association rate (u and dissociation rate (k〇ff). The equilibrium dissociation constant (Kd) value is calculated as kcff/ k or affinity is determined by immobilizing Αβ!, peptide on SA wafer and measuring the binding kinetics of Fab to fixed Αβ^ο peptide of 129629.doc •92- 200900079 9TL Fab and 9TL variant The affinity of the 9TL Fab fragment and its variant Fab fragment was determined by surface plasmon resonance (SPR) system (BIAcore 3000τμ, BIAcore, Inc., Piscaway, NJ). SA wafers were used according to the supplier's instructions (antibiotic) Streptavidin. The biotinylated Αβ peptide 1-40 was diluted in HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaC 3 mM EDTA, 0.005% P20) at 0.005 mg/ The concentration of mL was injected onto the wafer. Two ranges of antigen densities were achieved using variable flow times through individual wafer channels: 10-200 reaction units (RU) for detailed kinetic studies, and for concentration studies and For screening purposes, it is 500-600 RU. Regeneration Research Show 100 mM phosphoric acid (may also be followed by a solution containing 2 volumes of 50 mM NaOH and 1 volume of 70% ethanol) to effectively remove the bound Fab while maintaining the activity of the Αβ peptide on the wafer for more than 200 injections. HBS-EP buffer The solution was used as an electrophoresis buffer for all BIAcore assays. Serial dilutions of purified Fab samples (〇·1 -10x estimated KD) were injected at 1 〇〇pL/min for 2 min with a dissociation time of 10 min. ELISA and/or SDS-PAGE electrophoresis uses a standard Fab of known concentration (determined by amino acid analysis) to determine the concentration of Fab protein by fitting the data to a 1: Langmuir binding model (Karlsson, R Roos, H. Fagerstam, L. Petersson, B. (1994).

Methods Enzymology 6. 99-110) Using the BIAevaluation program to simultaneously obtain the kinetic association rate (k〇n) and the dissociation rate (k.^). The equilibrium dissociation constant (Kd) value is calculated as koff/kon. B. Antibody 9TL and variants thereof Αβ丨-binding affinity The amino acid sequence of the heavy and light chain variable regions of antibody 9TL is shown in Figure 1. 129629.doc -93- 200900079 The binding affinity of the 9TL antibody to Aβ 1-40 as determined using the two Biacore methods described above is shown in Table 2 below. Table 2. Binding affinity of antibody 9TL and Fab fragment k〇n (1/Ms) K〇ff(l/s) Κ〇(ηΜ) 9TL mAb on CM5 wafer, ΑβΜ0 flows on it 4.25 χ 105 3.89 χ ΙΟ-4 0.9 SA Wax on SAβι_40, 9TL Fab flowing on it 3.18 χ 105 3.59 χ ΙΟ '4 1.13 The amino acid sequence of the variant of 9TL is shown in Table 3 below. All amino acid substitutions of the variants shown in Table 3 are described relative to the 9TL sequence. The binding affinity of the Fab fragment of the 9TL variant is also shown in Table 3. KD and other kinetic parameters were determined by the above BIAcore analysis with Αβ!_4〇 immobilized on the SA wafer. Table 3. Amino acid sequence and kinetic data for antibody 9TL variants. Pure system Η1 (1) Η2 Η3 L1 L2 L3 k〇n (Ms1) (2) k〇ff (s'1) KD (nM) (3) 9TL 3.18xl05 3.59xl0'4 1.13 22-Τ/Ι L102I 3.18x10s 4.6〇xl O'4 1.45 C6 new L102T 3.56x10s 9.20X10-4 2.58 W1 Υ31Α, A34S L102T 3.18x10s 9.00x1 (T3 28.30 W8 Υ31Η, A34S, Κ35Α L102T 3.18x10s 3.80x10-3 11.95 W5 Υ31Η, Κ35Α L102T 3.18x10s 4.0〇xl〇·3 12.58 ΜΙ L94M 3.18xl05 8.60x10-4 2.70 M2 L94N 3.18x10s Ι.ΙΟχΙΟ'3 3.46 M3 L94C 3.18x10s 1.30xl0'3 4.09 Μ4 L94F 3.18x10s 9.95 xlO'4 3.13 Μ5 L94V 3.18x10s 1.65X10 '3 5.19 Μ6 L94K 3.18x10s 4.1〇xl〇·3 12.89 Μ7 L94S 3.18x10s 6.0〇xl〇·3 18.87 Μ8 L94Q 3.18x10s 6.8〇xlO'3 21.38 129629.doc -94- 200900079 Pure HI (1) H2 H3 LI L2 L3 k〇n (Ms,1) (2) k〇ff (s'1) KD (nM) (3) M9 L94G 3.18xl05 7.8〇xl〇·3 24.53 M10 L94S 3.18x10s 8.3〇xlO'3 26.10 Mil G96S 3.18x10s 2.00xl0'3 6.29 M12 G96T 3.18x10s 3.3〇xlO'3 10.38 M13 T97S 3.18x10s 3.9〇x lO'4 1.23 M14 H98L 3.18x10s 1.60X10'3 5.03 M15 Y99P 3.18xl05 6.7〇xl O'4 2.11 M16 Y99A 3.18x10s 7.00ΧΗΓ4 2.20 M17 Y99W 3.18x10s 1.00x1 O'3 3.14 M18 Y99Q 3.18x10s 1.5〇xl〇· 3 4.72 M19 Y99M 3.18xl05 1.70xl0'3 5.35 M20 Y99S 3.18x10s 2.0〇xl〇·3 6.29 M21 Y99E 3.18xl05 5.00xl0'3 15.72 M22 V101L 3.18x10s 4.0〇xl〇·3 12.58 M23 V101K 3.18x10s 5.00x1 O' 3 15.72 M24 V101H 3.18xl05 6.00x1 O'3 18.87 M25 Y101T 3.18x10s 8.00xl0·3 25.16 M26 V101A 3.18xl05 9.00x10.3 28.30 M27 Y101E 3.18x10s 1.2〇xl O'2 37.74 M28 V101M 3.18x10s 1.4〇xl O' 2 44.03 M29 L102S 3.18x10s 7.6〇xlO·4 2.39 M30 LI 02 V 3.18x10s 6.8〇xl 0·4 2.14 M31 L99V 3.18x10s 1.00x1 O'2 31.45 M32 L99I 3.18x10s 2.00xl0'2 62.89 M33 Y100W 3.18x10s 6.3〇 xlO'4 1.98 M34 S101T 3.18x10s 8.00xl0'4 2.52 M3 5 S101G 3.18x10s 9.00xl0'3 28.30 M36 L102R 3.18x10s 9.00xl0'4 2.83 M37 LI 02 A 3.18x10s 9·2〇χ10·4 2.89 M3 8 LI 02 V 3.18x10s 1.5〇xlO'3 4.72 M3 9 L102S 3.18xl05 2.3〇xlO&quot;3 7.23 M40 L102T 3.18x10s 4.50x10—3 14.15 M41 L102Q 3.18xl05 l.OOxlO'2 31.45 M42 L102E 3.18x10s 1.50X10'2 47.17 M43 VI041 3.18xl05 3.00xl0'4 0.94 129629.doc -95- 200900079 Pure system 1 (1) Η2 Η3 L1 L2 L3 k〇n (Ms1) (2) k〇ff (s '1) Kd (nM) (3) Μ44 V104T 3.18x10s 3.00x10-3 9.43 Μ45 V104P 3.18x10s 1.5〇xlO'2 47.17 Μ46 V104C 3.18xl05 2.00x1 O'2 62.89 Μ47 V104Q 3.18x10s 2·0〇χ10·2 62.89 Μ48 V104S 3.18xl05 2.6〇xlO'2 81.76 Μ49 V104N 3.18xl05 2.60x10-2 81.76 Μ50 V104F 3.18x10s 2.7〇xl O'2 84.91 Μ51 Υ105Η 3.18xl05 8.60X10·4 2.70 Μ52 Y105F 3.18x10s 1.3〇xlO'3 4.09 Μ53 Y105W 3.18x10s 1.3〇xl O'3 4.09 Μ54 Y105S 3.18x10s 2.4〇xl 0'3 7.55 Μ55 Υ105Ι 3.18x10s 3.00xl0'3 9.43 Μ56 Y105V 3.18x10s 3.5〇xl〇·3 11.01 Μ57 Υ105Α 3.18x10s 3.9〇χ10_3 12.26 1- There are extended CDR CDR, including Kabat and Chothia CDR two persons. The amino acid residues are numbered in sequence. 2 = underlined by experimental measurement. Others are estimated to be the same as 9TL. The 3=KD value is calculated as Kc^k^ff/k. . . Example 2: Characterization of the epitope on the peptide to which the antibody 9TL binds To determine the epitope on the Aβ polypeptide recognized by the antibody 9TL, surface plasma resonance (SPR, Biacore 3000) binding assay was used. The Λβ丨-40 polypeptide coupled to Bio Peptide Services (CO) was immobilized on a streptavidin coated wafer (SA wafer). The Aβ antibody Fab fragment (5〇ηΜ) was bound to the immobilized 4〇 in the absence or presence of a different Kolo fragment of the Aβ peptide (10 mM from American Peptide Company Inc., CA). The amino acid sequences of Αβ〗, Αβ&quot;2 and Αβ卜43 are shown in Table 4 below. The Αβ peptide that replaces the 9TL Fab fragment of the antibody with Αβ and the shoulder is Αβ28 4〇, 129629.doc •96-200900079 Αβ Bu 40, Αβ33-40 and Αβΐ7-40 (Fig. 2). Therefore, the antibody 9TL binds to the C-terminal peptide (33-40) of Αβι.4〇. As shown in Fig. 2, Αβι_28, Αβ28.42, Αβ22·35, Αβΐ·16, Αβι_4 3 and Αβΐ-38 did not inhibit the binding of the antibody 9TL Fab fragment, indicating that the antibody 9TL binds to the C-terminal end of the Αβι_4. In addition, Αβ 28-42 and Αβ 1 -43 do not inhibit the binding of antibody 9TL to Αβ 1 _4〇, although it can easily inhibit Αβ,-40 and control antibodies (antibody 2289, in US Application Publication No. 2) The binding of this antibody is described in 4/〇 146 512 and in WO 04/〇32808, which binds to Αβ!, 16-28. These results show that antibody 9TL preferentially binds to APi_4G but does not bind to Ap!-42 and Αβ!_43. Table 4. Amino acid sequence of beta-type amyloid peptide 1-40 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVV (SEQ ID NO: 15) 1-42 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA (SEQ ID NO: 16) 1-43 (WT) DAEFRHDSGYEYHHQKLVFFAEDVGSNKGAIIGL MVGGVVIAT (SEQ ID NO: 17) Example 3. Production of monoclonal antibody 2Η6 and deglycosylated 2Η6. Production and characterization of monoclonal antibody 2Η6 at 25-100 pg in adjuvant at about 16 consecutive weeks (each foot) The peptide (50 μΐ of the footpad), which is conjugated with KLH (amino acid 28-40 of Αβ丨-40) in a total of 100 μΐ per mouse, immunizes mice, as described in the following literature: Geerligs HJ et al. 1989, J. Immunol. Methods 124: 95-102; Kenney JS et al, 1989, J. Immunol. Methods 121: 157-166; and Wicher K et al, 1989, Int. Arch. Allergy Appl. Immunol. 89: 128-135. Mice were first immunized with 50 pg of peptide in CFA (complete Freud's 129629.doc -97-200900079 adjuvant). After 21 days, mice were immunized a second time with 25 tons of peptide in ifa (Fu's incomplete adjuvant). Twenty-three days after the second immunization, a third immunization was performed with 25 tons of peptide in IFA. Ten days later, the ELISA was used to test the antibody titer. After 34 days of the third immunization, a fourth immunization was performed with 25 gg peptide in IFA. After 32 days of the fourth immunization, final booster immunization was performed with 100 jig soluble peptide. Spleen cells were obtained from immunized mice and fused with NSO myeloma cells at a ratio of 1 : 1 with polyethylene glycol 15 。. The plated 〇ut hybrid was seeded in a 96-well plate in DMEM containing 2% horse serum and 2_grass acetate/pyruvate/insulin (Sigma) and started hypoxanthine/ Aminopterin/thymidine selection. On day 8, 1 μM of DMEM containing 20% horse serum was added to all wells. The supernatant was screened by using an antibody capture immunoassay. The determination of the antibody class is performed with a class-specific secondary antibody. A series of monoclonal antibody producing cell lines were selected for characterization. A selected cell line produces an antibody such as designated 2H6. This antibody was assayed to have an IgG2b heavy bond. The affinity of the antibody 2H6 to Αβ〗 was measured. The monoclonal antibody 2H6 was purified from the supernatant of the fusion tumor culture using protein a affinity chromatography. The supernatant was equilibrated to pH 8. The supernatant was then loaded onto Protein A column MabSelect (Amersham Biosciences # 17-5199-02) equilibrated to pH 8 with pbs. The column was washed with 5 column volumes of PBS (pH 8). The antibody was eluted with 50 mM citrate-phosphate buffer (pH 3). The lysed antibody was neutralized with 1 M phosphate buffer (pH 8). The purified antibody was dialyzed against PBS. The antibody concentration was determined by SDS_PAGE using a murine mAb standard curve. 129629.doc -98- 200900079 2H6 Fab was prepared by proteolytic degradation of the 2H6 whole antibody papain using the Immunopure Fab kit (pierce #44885) and purified by flow through Protein A chromatography following the manufacturer's instructions. . The concentration was determined by SDS-PAGE and A280 using 10D = 0.6 mg/ml. The affinity of the 2H6 monoclonal antibody was determined using a BlAcore 3000TM Surface Plasma Resonance (SPR) system (BIAcore, INC, Piscaway NJ). One method of determining affinity is to immobilize 2H6 antibody on a CM5 wafer and measure the binding kinetics of the Αβ!-^ peptide to the antibody. The CM5 wafer was activated with Ν-ethyl-Ν'-(3-diamidinopropyl)-carbodiimide hydrochloride (EDC) and hydrazine-hydroxysuccinimide (NHS) according to the supplier's instructions. The 2Η6 monoclonal antibody was diluted in 10 mM sodium acetate (pH 4.0 or 5.0) and injected onto the activated wafer at a concentration of 0.005 mg/mL. Using variable flow times through individual wafer channels, antibody densities are achieved in the range of 1000-2000 reaction units (RU) or 2000-3000 reaction units (RU). The wafer was blocked with ethanolamine. The regeneration study demonstrated that the mixture of Pierce Dissolution Buffer (Product No. 21004, Pierce Biotechnology, Rockford, IL) and 4 M NACL (2:1) effectively removed the bound Αβ, while the peptide retained 2H6 antibody activity on the wafer for 200 cycles. More than one injection. HBS-EP buffer (0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) was used as the running buffer for all BIAcore assays. A serial dilution of the purified Αβυο synthetic peptide sample (〇.1-1 (^ estimated 1&lt;:〇) was injected at 111°/11 in 11〇〇01^11 and allowed a dissociation time of 10 min. Fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using the BIAevaluation program 129629.doc • 99- 200900079 Simultaneously obtain the kinetic association rate (U and dissociation rate (kQff). The equilibrium dissociation constant (KD) value is calculated as kcff / ku. Or, affinity is fixed on the SA wafer by measurement and measurement The binding kinetics of the 2Η6 Fab to the immobilized ΑβΝ4〇 peptide was determined by the surface plasmon resonance (SPR) system (BIAcore 3000TM, BIAcore, Inc., Pi seaway, NJ) to determine the affinity of the 2H6 Fab fragment. Instructions for use of SA wafers (streptavidin). Biotinylated Αβ peptide 1-40 (SEQ ID NO: 15) was diluted in HBS-EP (10 mM HEPES (pH 7.4) &gt; 150 mM NaCl &gt ; 3 mM EDTA &gt; 0.005% P20) and injected onto the wafer at a concentration of 0.005 mg/mL. Flow time to achieve two ranges of antigen density: 10-200 reaction units (RU) for detailed kinetic studies and 500-600 RU for concentration studies. Regeneration studies show Pierce Dissolution Buffer with 4 M The mixture of NaCl (2:1) effectively removed the bound Fab while maintaining the Αβ peptide activity on the wafer for more than 200 injections. HBS-EP buffer was used as an electrophoresis buffer for all BIAcore assays. Will it be purified? Serial dilutions of the 813 samples (0.1-10&gt;&lt;1^13) were injected at 10〇41^/111111 for 2 min and allowed to dissociate for 10 min. by ELIS A and/or SDS-PAGE electrophoresis The concentration of Fab protein is determined by standard Fab at a known concentration (determined by amino acid analysis) by fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L.). Petersson, B. (1994). Methods Enzymology 6. 99-1 10) Use the BIAevaluation program to simultaneously obtain the kinetic association rate (kon) and the dissociation rate (k). ^). The equilibrium dissociation constant (KD) value is calculated as Iff/k. . . The affinity of the 2H6 antibody determined using the above two methods is shown in Table 5 129629.doc -100-200900079 below. The affinity of the murine antibody 2286 which binds to the peptide of the amino acid 28-40 of Αβ^ο was tested as described above. Antibody 2286 is described in U.S. Application Serial No. 10/683,815 and PCT/US03/32080. Table 5. Binding affinities of antibodies 2Η6 and 2286 k〇n (1/Ms) K〇ff(l/s) KD (nM) 2Η6 mAb on CM5 wafer, Αβ丨-40 flows above 4.67 χ 105 3.9 χ 103 9 ΑβΜ〇 on the SA wafer, 2Η6 Fab flowing on the above 6.3 χ 10s 3.0 χ 10'J 4.7 CM5 wafer 2286 mAb, Αβ]_4〇 flowing above 1.56 χ 105 0.0419 269 SA on the wafer Αβι_4 〇, 2286 Fab flows above 1.8 χ 10s 0.044 245 To determine the epitope on the Αβ polypeptide identified by antibody 2Η6, surface plasmon resonance (SPR, Biacore 3000) binding assay was used. A Αβ Bu 40 polypeptide (SEQ ID NO: 15) coupled to Bio Peptide Services (CO) was immobilized on a streptavidin coated wafer (Sa wafer). The Αβ antibody (100 nM) was bound to immobilized Αβ! in the absence or presence of a different soluble fraction of Αβ peptide (16 μΜ from American Peptide Company Inc., CA). The Αβ peptides that replace the binding of antibody 2H6 to Αβ, -40 are Αβ17·40, Αβ33_40 and Αβ, Μ Figure 3). Therefore, the antibody 2Η6 binds to the C-terminal peptide (33-40) of Αβ 丨-40. However, Cβ, the C-terminal peptide (33-40) did not bind to antibody 2286 at the test concentration. As shown in Figure 3, the peptide does not inhibit binding of antibody 2Η6 or antibody 2286 to Αβ^, indicating that similar to antibody 2286, the epitope bound by antibody 2Η6 includes the amino acid 39 of the Αβ__40 peptide and/or 40 (Figure 3). In addition, Αβυζ and peptide do not inhibit the binding of antibody 2Η6 to APwo, although it is readily inhibited by Αβ^ο and the control antibody (antibody 2289, described in US Application Nos. 10/683,815 and PCT/US03/32080) 129629.doc -101 - 200900079 Conclusion =, this control antibody binds to 16_28 of 4_4〇 (Figure 3). These results show that the antibody 2H6 binds preferentially to Αβ丨, but does not bind to ΑβΜ2 and Αβ丨. In order to improve the involvement of the discrete amino acid residues of the β-amyloid peptide bound by the antibody 2Η6, II variants were induced by site-directed mutagenesis, of which the last 6 amino acids (Αβι, amino acid residues 35_4) Each of the 〇) was replaced by alanine (induced by alanine scanning mutation). These variants (sequences shown in Table 6) were expressed in E. coli as a glutathione-s-transferase (GST) fusion protein (Amersham sinensis Bioteeh' Plscataway, NJ USA)' Affinity purification was performed on glutathione-agarose beads (Sigma-Aldrich Corp., St. Louis, MO, USA). As a control, 'wild type (wt) A|3i_4() and Α(3ΐ4, Αβυζ and Αβ〗_39 also appeared as GST fusion protein. Then α(3ι 4(), ΑβΝ41, Αβ]_42, Αβ!·39 And six different variants (1^35 eight (1-40), V36A (l-40), G37A (l-40), G38A (l-40), V39A (1-/ 40) shown in Table 6. ), V40A (l-40)) fixed (100 μl 0.025 pg/ml GST peptide/well) on ELISA assay plate 'and down from 0.3 nM (shown in Figure 4 using 0.3 nM mAb) The serial dilutions were incubated with either of mAbs 2286, 2289, and 2H6. After 1 consecutive washes, the assay plate was sequentially passed through 100 μΐ 〇.〇3 pg/ml of biotin-conjugated goat anti-small. Mouse (h+L) antibody (Vector Laboratories, vector #BA-9200, Burllingame CA, USA), HRP-conjugated streptavidin (Amersham Biosciences Corp., #RPN4401V, NJ, 100 μl 0.025 pg/ml per well) USA) Incubation. Read the absorbance of the plate at 45 0 nm. 129629.doc -102· 200900079 Table 6. Amino acid sequence of beta-type amyloid peptides and variants 1-40 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGYV (SEQ ID NO: 15) 1-42 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA (SEQ ID NO: 16) 1-43 (WT) DAEFRHDSGYEVHHQKLVFFAEDYGSNKGAIIGL MVGGVVIAT (SEQ ID NO: 17) 1-41 (WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVI (SEQ ID NO: 18) 1-39 (WT DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGV (SEQ ID NO: 19) Μ35Α(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL AVGGVV (SEQ ID NO:20) V36A(l-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MAGGVV (SEQ ID NO:21) G37A(l-40) DAEFRHDSGYEVHHQKLYFFAEDVGSNKGAIIGL MVAGVV (SEQ ID NO: 22) G38A (l-40) DAEFRHDSGYEVHHQKLVFFAEDYGSNKGAIIGL MVGAVV (SEQ ID NO: 23) V39A (l-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGAV (SEQ ID NO: 24) V40A (l-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVA (SEQ ID NO: 25) As shown in Figure 4, Mab 2289 for amino acids 16 to 28 of Αβ identified all variants with the same intensity and served as an internal positive control for protein concentration and protein integrity on the plate. As shown in Figure 4, antibody 2H6 did not recognize ApU41, Αβ "39 or Αβ卜42. Αβι_4〇 variants V40A, V39A, G38A, G37A, V36A and Μ35Α exhibited binding to antibody 2h6, demonstrating antibody 2H6 antigen The determinant base extends at least 6 amino acids at the C-terminus of Αβ!. The mutations of V and G to Α are extremely conservative mutations and are unlikely to produce important conformational changes in the protein. Therefore, these mutations are directed against antibody 2H6. The greater effect of binding can be attributed to the ability of the antibody to distinguish between the mentioned amino acids in the AyS range, and such data demonstrate the extremely high degree of specificity of this antibody. To determine if 2H6 and 9TL are associated with Aβ 1- 40. Competition and competition, using the Biacore assay for competition experiments. Antibody 2Η6, 9TL and 2289 固•103·129629.doc 200900079 were placed on different channels of the CM5 wafer. According to the supplier's instructions with N-ethyl W-( 3-diaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activate CM5 wafer channels. Antibody 2H6, 9TL and 2289 are each diluted in 10 mM sodium acetate (pH 4.0) and injected at a concentration of 0.005 mg/mL for activation On-chip. Antibody density was 1625 reaction units (RU) for 2H6; 4000 RU for 9TL; and 2200 RU for 2289. Block each channel with ethanolamine. Αβ丨_4〇 peptide (150 μΜ) Flowing on the wafer for 2 min. Then 0.6 μΜ of antibody 2Η6 (to be tested for binding competition) was flowed on the wafer for 1 min. HBS-EP buffer (0.01 M HEPES (pH 7.4), 0·15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20) was used as an electrophoresis buffer for all BIAcore assays. Measured by binding of Αβ!_4〇 by Pierce Dissolution Buffer (Product No. 21004, Pierce Biotechnology, Rockford) , IL) and a mixture of 4 Μ NaCl (2:1) were washed twice for 6 sec to regenerate all channels of the wafer. Next, the antibody 9TL was followed by competitive binding to antibody 2289. It was observed that between 9TL and 2H6 and Αβυ. Binding competition, but no competition was observed between 9TL and 2289 or between 2Η6 and 2289. The observations of competition between immobilized antibodies and the same antibodies flowing on the wafer served as a positive control. 抗体. Antibody 2Η6 Not combined with ΑΡΡ for determination 2Η6 No binding to the amyloid precursor protein (ΑΡΡ), binding was measured 2Η6 ΑΡΡ wild type transfected cells. In the precursor protein of cDNA encoding wild type human amyloid-transfected into 293 cells. After transfection for 48 hours, the cells were incubated for 45 minutes on ice with monoclonal antibody anti-ΑβΜ6, anti-Αβ16.28 or 2H6 (5 129629.doc -104-200900079 pg/mbu in DMEM with 10% FCS). The cells were then washed three times in PBS for 5 minutes and fixed with 4% PFA. The cells were washed three more times in PBS and antibody binding was detected from a secondary Cy3-conjugated goat anti-mouse antibody (1:500 dilution) from Jackson Immunoresearch under a fluorescent microscope. Both the N-terminal or central epitope-determining agent in Αβ and the anti-Αβ16.28 antibody both exhibited significant binding to the scorpion precursor protein expressed on the cell. In contrast, 2Η6 does not bind to sputum expressing cells. C. Deglycosylation antibody 2Η6 was produced to produce deglycosylated antibody 2Η6, peptide-N-glycosidase F (Prozyme, 0.05 U) in 20 mM Tris-HC1 (pH 8.0) at 37 °C /mg antibody) The purified antibody 2H6 was incubated for 7 days. The integrity of deglycosylation was tested by MALDI-TOF-MS and protein gel electrophoresis. The deglycosylated antibody was purified by protein A stratification and the endotoxin was removed by Q-agarose. The Biacore assay described above was used to test the binding affinity of deglycosylated 2H6 to Αβ, ^ο, and the deglycosylated 2Η6 pair Αβ! was found to have a binding affinity to the intact antibody 2Η6. Example 4: Role of deglycosylated antibody 2H6 (2H6-D) in protecting and restoring retinal function in an animal model of age-related macular degeneration

The initial study (Malek, G. et al., PNAS 102: 1 1900-5 (2005)) demonstrated that the combination of the following three risk factors produced an animal model that closely resembled the clinical features of human AMD: (1) Lipoprotein isoforms E4 (APOE4) genotype, (2) old age (65 weeks and older) and (3) high fat and cholesterol rich (HF-C) diet. Old APOE4 mice develop lesions similar to the morphological features observed in dry and wet human AMD 129629.doc -105-200900079, including retinal pigment epithelial (caffe) pigmentation, #diffusion RPE deposition, month The target area is rich in choroidal sputum deposition, Bruch's membrane thickening, RpE atrophy of the overlying photoreceptor degeneration, and choroidal neovascularization (CNV) e, regardless of the dietary regimen. None of these changes were detected in either of them, nor was any pathology detected in young animals. The full field sc〇t〇pic electr(10)tin〇gram was identified as a functional deficiency. The affected animals had a significant decrease in a-wave amplitude and b-wave amplitude compared to the control. It is important that all three risk factors exist for these histopathological and functional changes. The animal model of spontaneous occurrence of cnv first has physiologically relevant risk factors for human diseases. A.t test plan clothing: invited to teach #. Old-age (65-week-old) dry-replacement mice expressing human ApoE4 were used for the experiment. It has been previously disclosed (Maiek, g. et al., PNAS 102: 1 1900-5 (2005)) that there is an AMD-like phenotype in these mice. For the eight-week treatment study, a 65-week-old or 65-week-old Ap〇E4 transgenic mouse was assigned to one of the four groups. The first group (E4_ND) was kept in a normal diet („=:2). The second group (E4_HFC_R1) was fed a high-fat, cholesterol-rich (HF-C) diet for 8 weeks (w=2). Three groups (E4_HFC-R1) were fed the HF-C diet for 8 weeks and received an intraperitoneal injection of Rinat 1 (3 mg/kg) per week („ = 5). The fourth group (E4-HFC-R2) was fed the HF-C diet for 8 weeks and allowed to receive a weekly Rinat 2 (3 mg/kg) intraperitoneal smear (/75). After the study of Nine-Yuancheng, unmasked for each group: Rinat 1 is a PBS vehicle and Rinat 2 is a deglycosylated anti-Aβ antibody 2H6 (as described in Example 129629.doc • 106- 200900079 Example 3) Individual anti-human Αβ28·4〇IgG2b, 2H6-D,). Live invitations are well-defined. Fundus examination was performed at week 28, and fundus and luciferin angiography were performed at week 8. Use a fundus camera (TRC-50EX retina camera) to take photos. Capture images using the TOPCON IMAGEnetTM system.萤 Fluorescein dye (10% luciferin sodium, about 0.1 mL/kg) was injected from the vascular access p〇rt. After the dye injection, photographs were taken at several time points to include the arterial phase, the early arteriovenous phase, and several late arteriovenous phases&apos; in order to assess neovascularization and monitor luminescence leakage associated with CNV lesions. Interpretation and analysis of luciferin angiography was performed independently by an ophthalmologist. Total plasma cholesterol levels in whole blood were collected from mice (fasting for 5 hours) before and after the 8-week HF-C diet. Luxury Dragon Cover #靡影肄. One animal (E4-HFC-R2) exhibited possible leakage in the late framework of angiography. After F a but before the electroretinogram, the animal died and no tissue was recoverable.祎 腐 腐 羼 。 record. During the ninth week, electroretinogram (ERG) recordings were obtained from animals that were dark adapted for at least 12 hours. Each animal was anesthetized with a mixture of chloramine _/ phenothiazine, the pupil of which was enlarged and stabilized at 37 in the animal. (After warming the pad, use the same drop of 2.5% hydroxypropyl methylcellulose placed on the silver wire test electrode in contact with the eye to record the ERG trace. Place the mouse in a light-sensitive stimulation chamber where the animals are placed Exposure to light flash (fIashes 〇f hght) (maximum intensity 1000 cd_s/m2, decay from ^ logarithmic step length from 〇〇〇〇5). Measurement of a-wave amplitude from baseline to a-wave valley, and from a Bobo Valley to b wave crest measures the b-wave amplitude. 129629.doc -107· 200900079 Yutiefen#. On the day of killing, the mice were weighed and over-administered with Avenin (0·2 μl/10 gm body weight) ), and then perfused intracardiac with 2 mL of saline. Quickly remove the brain' and immerse the left half of the brain in freshly prepared for histopathology. h. Pre-treat a 30 micron vibrating section (vibratome section) with 1% MeOH, 1x PBS, and 2% Η&quot;2 'wash it in PBS, incubate in 88 〇/capric acid for antigen Recovered and blocked with 5% normal goat serum (Ngs) and pbs. 4G8 primary antibody was labeled with biotin in 1% NGS/PBS ( The 1:1000 dilution of Signet 4G8 monoclonal mouse IgG2b) of the amino acid residues 17-24 of the beta-based starch was incubated overnight and was performed using the ABC Vectastain kit (Vector Labs) as described by the manufacturer. Visually. B. Results Recover/protect retinal function by administering deglycosylated antibodies. As shown in Figure 5, compared to mice in normal diet (E4_ND), fat and cholesterol-rich diets in fake foods In the Ap〇E4 mouse (E4-HFC), there is a statistically significant decrease in the amplitude of the a wave and the amplitude of the b wave (a wave p-0.0106 'b wave p=〇.〇〇8). The obtained erg was compared with the baseline set of ERG. Compared with E4_HFC (not shown), there is no significant difference in the amplitude of a wave in any of the injected groups (E4_HFC R1 &amp; E4_HFC_R2). Conversely, In the E4-HFC-R2 group, the b-wave amplitude exhibited a striking recovery and/or protection of retinal function (Fig. 6). The deposition of anti-inhibition antibody 2H6_DiAp〇E4 in the HF-C diet was reduced. As illustrated in Figure 7, after 8 weeks of immunotherapy with antibody 2H6-D' compared to the control vehicle group E4-HFC mice in total Αβ 129629.doc •108· 200900079 plague staining decreased. C ·In conclusion, the above data prove: 丨) recovery / protection of retinal function, such as by injection of anti-Αβ antibody 2H6-D The ERG of the mouse was demonstrated, and 2) compared to the untreated AMD mouse group, when the antibody 2H6-D was treated in the mouse brain, the deposition of the powder was reduced. Example 5. Determination of binding affinity of antibody 6G and its variants A. General Methods The following general methods were used in this and other examples. Fab fragment expression for expression vector antibodies for pure line characterization is initiated as described in Barbas (2001) corpse display: a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press pg 2.10. Vector pComb3X) Under the control of the IPTG-inducible lacZ promoter, 'however, the modification includes the addition and expression of the following additional domains: human kappa light chain constant domain of IgG2a human immunoglobulin and CHI constant domain, ig γ_2 chain C region, protein accession number 01 859; Immunoglobulin kappa light chain (Homo sapiens), protein accession number CAA09181. Small scale Fab preparation Fab small scale performance in 96 well plates was performed as follows. Starting with E. coli transformed from Fab library, colonies were selected to inoculate mother plates (agar LB + ampicillin (50 gg/ml) + 2 ° / 〇 glucose) and working plates (2 ml / well, 96 well / plate) Contains 1.5 mL LB + ampicillin (50 pg/mi) + 2% glucose). Both plates were grown for 8-12 hours at 30 C. The mother plate was stored under 4X: and the cells from the working plate 129629.doc -109- 200900079 were granulated at 5000 rpm and re-slurried with 1 mL LB + ampicillin (5 〇pg/ml) + 1 mM IPTG. Suspended to induce Fab expression. The cells were harvested by centrifugation after 5 h of performance time at 3 ° C, and then resuspended in 500 pL of buffer HBS-P (10 mM HEPES buffer (pH 7.4), 150 mM Naa, 0.005% P20). )in. Lysis of hbs-ep resuspended cells was achieved by one cycle of freezing (-80 °C) followed by melting at 37 °C. The cell lysate was centrifuged at 5000 rpm for 30 min to separate cell debris from the supernatant containing the Fab. Next, the supernatant was injected into the BIAcore plasma resonance device to obtain the affinity information of each Fab. The pure line of Fab was rescued from the mother plate to sequence the DNA and was used for large scale Fab manufacture and detailed characterization as described below. Large scale Fab preparation To obtain detailed kinetic parameters, the Fab was expressed and purified from large cultures. A 5 mL overnight culture from the selected Fab-expressing E. coli strain was used to inoculate a conical flask containing 200 mL of LB + ampicillin (5 〇 gg/ml) + 2% glucose. The pure line was incubated at 3 (TC) until D5Qnm was reached, and then induced by substituting the medium for 2 〇〇 with LB + ampicillin (5 〇 Kg/ml) + 1 mM IPTG. After the performance time of Ct5 h, the cells were granulated by centrifugation, and then resuspended in PBS (pH 8) by freezing/thawing (under -8 〇 and 37 37, respectively) The two 楯% of the cells were lysed by the cells. The cell lysate was loaded on a Ni_NTA super-flow agarose (1) iagen, Vai (such as CA) &amp; column, which was equilibrated with PBS^pH δ), followed by 5 The column volume is 8) washed. Individual Fabs were dissolved in different fractions with pBS (P 8) 300 mM imidazole. Fractions containing Fab containing 129629.doc •110-200900079 were pooled and dialyzed against pR&lt;5&gt;&amp;&amp;&gt;, followed by ELISA, followed by affinity characterization. Whole antibody preparation To express whole antibodies, the heavy and light chain variable regions were cloned in mammalian expression vectors and transfected into HEK 293 cells using lipofectamine for transient expression. Protein A was used to purify the antibody using standard methods. The vector pDb.6G.hFc2a is a expression vector comprising a heavy chain of a 6G antibody and is suitable for transient or stable expression of a heavy chain. Vector 15] 〇13.6(}.}11^2&amp; has a nucleotide sequence corresponding to the following region: murine cell giant viral promoter region (nucleotides 1-612); synthetic intron (nucleotide 619_15〇7) ; DHFR coding region (nucleotide 707-1167); human growth hormone signal peptide (nucleotide 1525_ 1602); heavy chain variable region of 6G; human heavy chain IgG2a constant region containing the following mutation: A330P331 To S330S331 (refer to the wild type 1 § (} 2 &amp; sequence amino acid number; see Eur. J. Immunol. (1999) SV40 late polyadenylation signal; SV40 enhancer region; 喔 体 fl fl region and β The purine aminotransferase (AmpR) coding region. The vector pEb.6G.hK is a expression vector containing the light chain of the 6G antibody and is suitable for transient expression of the light chain. The vector pEb.6G.hK has a nucleotide sequence corresponding to the following region : murine cell giant virus promoter region (nuclear acid j _ 612); human EF-1 intron (nucleotide 619-1142); human growth hormone signal peptide (nucleotides 1 173-1 150); Antibody 6G light chain variable region; human ulnar bond constant region; SV40 late polyadenylation signal; SV40 fortifier region; sputum f-region and beta-prolinease (AmpR) Code area.

Biacore Assay 129629.doc -111 - 200900079 The affinity of 6G monoclonal antibodies was determined using the BlAcore 3000TM Surface Plasma Resonance (SPR) system (BIAcore, INC, Piscaway NJ) using the method described in Example 1 above. ELISA assay ELISA was used to measure binding of antibody 6G and variants to non-biotinylated Aβ peptide. The NUNC maxisorp plate was coated with 2.5 pg/ml Αβ peptide in PBS (pH 7.4) for more than 1 hour at 4 °C. Plates were blocked with 1% BSA in PBS buffer (pH 7.4). Primary antibodies (from cell supernatant, serum containing anti-Aβ antibody or purified whole antibody or Fab at the desired dilution) were incubated with the immobilized Aβ peptide for 1 h at room temperature. After washing, the plates were incubated with secondary antibodies (HRP-conjugated goat anti-human kappa chain antibody (MP Biomedicals, 55233) diluted 1:5000). After washing, the bound secondary antibody was measured by adding TMB receptor (KPL, 50-76-02, 50-65-02). The HRP reaction was terminated by the addition of 1 guanidine phosphate and the absorbance at 450 nm was measured. ELISA was used to measure binding of antibody 6G and variants to biotinylated Aβ peptide. The NUNC maxisorp plate was coated with 6 pg/ml streptavidin (Pierce, 2 1122) in PBS (pH 7.4) for more than 1 hour at 4 °C. 1 ° / in PBS buffer (pH 7.4). BSA to block the board. After washing, the biotin-labeled Aβ peptide in PBS (pH 7.4) was incubated for 1 hour at room temperature. The primary antibody (from the cell supernatant, the serum containing the anti-Aβ antibody or the purified whole antibody or Fab at the desired dilution) was incubated with the immobilized Aβ peptide for 1 h at room temperature. After washing, the plates were incubated with secondary antibodies (HRP-conjugated goat anti-human kappa chain antibody (MP Biomedicals, 55233) diluted 1:5000). Washing 129629.doc -112- 200900079 After washing, the bound secondary antibody was measured by adding TMB receptor (KPL, 50-76-02, 50-65-02). The HRP reaction was terminated by the addition of 1 guanidine phosphate and the absorbance at 450 nm was measured. B. Binding Affinity of Antibody 6G and Variants to Aβ, Αβ!_42 and Other Aβ Peptides The amino acid sequences of the heavy and light chain variable regions of antibody 6G are shown in Figure 8. The binding affinities of the 6G antibodies measured using the above Biacore to ΑβΝ40, Αβ^ and Αβ22.37 are shown in Table 7 below. Table 7. Binding affinity of antibody 6G Fab fragment k〇n (1/Ms) k〇ff(l/s) KD (nM) Biotin-labeled APmg immobilized on streptavidin wafer, 6G Fab flows On it, 3.0X105 7.〇χ10'4 2 was immobilized on the biotin-labeled biotin-labeled Αβι-42, and the 6G Fab was flowed on it. 1.8xl04 1.6xlO'J 80 was immobilized on the streptavidin wafer. The biotin-labeled Αβ22-37, 6G Fab flows over it 3.6 χ 105 3.9 x 10 'j 11 The amino acid sequence of the 6G variant is shown in Table 8 below. All amino acid substitutions of the variants shown in Table 8 are described relative to the sequence of 6G. The relative combinations of the 6G variants are also shown in Table 8. Binding was determined by the above ELISA using a non-biotin label or Αβ, Α immobilized on the surface of the ELISA plate. Table 8. Amino acid sequence and binding data for antibody 6G variants. 6G heavy chain mutant variant binding data obtained by ELISA Α450 ELISA pure line number mutation Αβΐ-40 Αβι_42 6G F99 D100 Ν101 Υ102 D103 R104 2.55 0.95 1A Y 1.60 0.26 1B Μ 0.37 0.22 1G L 0.51 0.21 2G Ρ 0.30 0.50 3E C 0.26 0.40 4G S 1.41 0.30 5D Ν 1.52 0.39 6A Τ 0.86 0.31 129629.doc -113 - 200900079 7Β S 0.44 0.27 7D C 0.23 0.31 8Η Η 0.21 0.19 9Ε R 0.22 0.26 10Α F 1.85 0.34 10Ε L 0.41 0.24 10G I 0.63 0.22 11D M 0.29 0.24 2F P 1.89 0.38 3Α A 1.16 0.28 3Β R 1.43 0.43 3C G 2.30 0.76 4Α G 2.17 0.40 4Β F 2.48 0.71 4D 0 2.45 1.00 6F S 2.28 0.62 6G light chain mutant variant binding material A450 by ELISA ELISA pure line number mutation Αβΐ-40 Αβΐ-42 6G 〇93 Q94 S95 K96 E97 F98 P99 W100 S101 2.49 0.61 2H K 0.07 0.13 3A P 0.08 0.13 4F S 2.00 0.30 5B G 0.09 0.14 7E R 0.09 0.18 7F K 0 .12 0.19 10E L 0.08 0.12 1A N 2.02 0.32 1C F 0.05 0.05 4A A 2.09 0.28 4G F 1.07 0.28 5H R 2.60 0.85 6C G 0.05 0.05 6D T 2.41 1.34 6E P 0.12 0.20 8G V 2.60 0.90 Example 6: Antibody 6〇 Characterization of the epitope on the conjugated Ap peptide is to determine the epitope on the Αβ peptide recognized by antibody 6G, using ELIS assay. Various Αβ peptides (Global Peptide Services, C〇) were immobilized on an ELISA plate. Binding of 6G whole antibody (20 nM) to immobilized Ap was determined by ELISA as described above. The amino acid sequences of Api 4〇, 丨-42 and Αβ1·43 are shown in Table 9 below. As shown in Figure 9, antibody 6G and Αβ 129629.doc -114- 200900079 peptides 17-40, 17-42, 22-35, 28-40, 1-38, 1-40, 1-42, 1-43 And 28-42 binding; but the combination with 28-42 is much weaker than the binding of other Αβ peptides. Antibody 6G does not bind to Aβ peptides 1-16, 1-28 and 33-40. Thus, antibody 6G binds to the C-terminus of various truncated Aβ peptides such as 22-35, 1-3 8, 1-40, 1-42 and 1-43. Table 9 below shows a comparison of the binding affinity of 6G versus ΑβI measured by koff(l/s) with other Αβ peptides as measured by Kia(1). Antibody 6G has the highest affinity compared to other peptides in combination with Αβ!, which has a significantly lower affinity for the short Αβι·4() (such as 1-36, ^7, ^ recognition, Αβι·4ΑΑρΐ43). This indicates that the side chain or main chain of the amino acid 40 (proline) of Αβ is related to the binding of 6G to Αβ!; and the binding is significantly reduced when the amino acid is absent (for example, about 10 to about 50-250 fold loss of affinity). The combination of lower affinity and sulfhydrylation of the sulfhydryl group indicates that the binding involves (but does not depend on Μβ), the free c-terminus. Αβ丨42&amp;ββΐ 43 The lower affinity binding can be attributed to the difference in configuration between the monomeric forms of 八131_4() and 八1_42 or Α(3ΐ43. The monomer of Αβ-42 has been shown to be different from Αβι-4〇 in solution. The configuration of the monomer. See the monomeric structure equivalent of the accession number 1ΙγΤ2 Αβι·42 shown in the Pr〇tein Data Bank (Pdb file); and the protein library (pdb file) The monomer structure equivalent of Αβ! of the accession numbers 1BA6 and 1BA4. 129629.doc -115- 200900079 Table 9 Αβ peptide fragment k Ff(l/s) Κ〇ίϊΑβ peptide/Ι^Αβ!^) (Affinity loss multiple) 1-28 - 1-43 Very low binding 22-35 0.0285 215.9 1-36 &quot;0.0205 155.3 1-37 0.0149 112 8 1-38 9.3 X ΙΟ'3 70.4 1-39 7.92 X 10—J 60.0 17-42 0.0465 352.2 1-42 1.9 χ 1 (TJ 14.4 28-42 —3.37 χ 10. ~ 25.5 28-40-NH2# — 3.62 χ 10” 27.4 28-40 —6.4 χ 1 (T4 4.8 17-40 — 2.15 χ 10·4 ～ 1.6 1-40 1.32 χ ΙΟ·4 — 1 As an analyte, the peptide flows with a fixed chemical by amine 6 〇 monoclonal antibody (ligand) on the CM5 wafer #carboxy-terminal amidated peptide The epitope of antibody 6G was determined by ELIS A assay. Various Αβ peptides were added (the peptides were added to the C-terminus) Biotin-labeled 15-mer (15-mer) or 10-mer (l-mer) was immobilized on streptavidin-coated plates. Antibody 6G (2.5 pg/ml was immobilized with immobilized peptide) Up to 10 pg/ml) and binding as described above. As shown in Figure 1, antibody 6G with amino acids 20-34, 21-35, 22-36, 23-37, 24-38, 25 -39 and 25-34 Αβ peptide (having glycine at the C-terminus) binding; However, it does not bind to an Αβ peptide having amino acids 19-33, 26-40, 27-41, 24-33 and 26-35 (having glycine at the terminal C of these peptides). This indicates that the epitope to which antibody 6G binds includes amino acids 25 to 34. Based on the information shown above, the epitope to which the antibody binds to 6G appears to contain 129629.doc-116-200900079 comprising amino acids 25-34 and 40. Figure 11 is a schematic diagram showing the epitope of antibody 6G. B. Antibody 6G does not bind to APP To determine whether 6G binds to a starch-like precursor protein (APP), binding of 6G to cells transfected with wild-type APP is determined. HEK293 cells were transfected with a cDNA encoding a wild-type human-type starch precursor protein. After transfection for 48 hours, the cells were incubated for 45 minutes on ice with monoclonal antibody anti-ΑβΜ6, (m2324) or 6G (5 pg/ml in DMEM with 10% FCS). The cells were then washed three times in PBS for 5 minutes and fixed with 4% PFA. The cells were washed three more times in PBS and antibody binding was detected from a secondary Cy3 junction goat anti-mouse antibody (1:500 dilution) from Jackson Immunorese arch under a fluorescent microscope. As shown in Figure 12, the anti-antibody display that recognizes the N-terminal epitope in Αβ exhibits significant binding to the scorpion precursor protein expressed on the cell. In contrast, 6G does not bind to sputum expressing cells. Example 7: Production and characterization of murine anti-sputum 7G10 (anti-APbu/APws) Mice were immunized with ~1 00 pg of peptide conjugated to KLH in an adjuvant, such as Konig, G. et al., Annals New York Academy Said in Sciences. 777:344-55 (1996). Since the position 29-42 of the BA4 peptide is completely within the APP putative transmembrane region and is hydrophobic in nature, the KLH peptide is joined to the hydrophilic spacer. KLH-HDGDGD-MVGGVVIA was synthesized under Anaspec and the 5 residue spacer was sufficient to overcome the insoluble problem and extend the C-terminus away from the carrier. On the first day, mice were immunized subcutaneously with 100 pg of 3 5-42/KLH peptide with CFA (Fuss Complete Adjuvant). Mice were immunized with 100 peptide/KLH Ribi/矾 on day 129629.doc -117 - 200900079. On day 55, mice were immunized as on day 15. On day 95, mice were boosted intraperitoneally at 100 pg peptide/KLH. Splenocytes were obtained from immunized mice and fused with P3x63Ag8.653 myeloma cells (Atcc CRL 1580) at a ratio of 10:1 on day 99 using polyethylene glycol 丨5〇〇 fusion. The fused cells were seeded in 96-well plates (p丨ate(} int〇) DMEM containing 2% horse serum and 2_grass acetate/pyruvate/insulin (Sigma) and The solution was started on the 10th day after fusion using the Elisa assay and coated with 2 pg/ml Αβ丨-42 (Anaspec). The positives were selected and amplified and further characterized. When tested Αβ〗 _42 free peptide The serum titer of the mice on the day of fusion was 1/9000. The affinity of 7G10 to Αβ1-40, 1-42 and 1-43 was analyzed by Biacore.

Biacore Assay The affinity of the 7G10 antibody was determined using BIAcore 3000® as previously described in Example 1. N-biotinylated Αβ1-40, 1-42 and 1-43 were captured on SA sputum. Starting from the 1 / 6 dilution of the 7 G1 0 stock, the three-fold dilution series of anti-Αβ1-40 Fab, anti-Αβ1-42 Fab and anti-Αβ1-43 Fab were injected. The wafer was regenerated with a 18 sec pulse of 6% EtOH + 6 mM NaOH. Sample IgG (mg/mL) Volume (mL) Fab (mg/mL) Volume (mL) KD(nM) Αβι·4〇peptide 0.672 2.4 1.458 0.4 Not applicable Αβι_42 Shape 0.672 2.4 1.458 0.4 37.6 ΑβΜ3 peptide 0.672 2.4 1.458 0.4 41 As indicated above, 7G10 has a KD of 37.6 nM for the ΑβΝ42 peptide and a KD of 41 nM for the peptide. The measurable binding was not detected for Αβ丨_4 〇 peptide. 129629.doc -118- 200900079 Example 10: Comparison of antibodies 9TL, 6G and 7G10 in protecting and restoring retinal function in age-related macular degeneration animal models A. Experimental protocol. The protocol as described in Example 4 above was repeated. Assigning ΑροΕ4 transgenic mice of 65 weeks of age or older to one of the groups for eight weeks. Keep the first group (E4-ND) in a normal diet 〇=6). The second group (E4-HFC-R1) was fed a high-fat, cholesterol-rich (HF-C) diet for 8 weeks (“12”). The third group (E4-HFCMu;)|^f HF-C diet was administered for 8 weeks and received an intraperitoneal injection of Rinat 3 (3 mg/kg) per week ("12"). The fourth group (E4-HFC-R2) was fed the HF-C diet for 8 weeks and received an intraperitoneal injection of Rinat 4 (3 mg/kg) per week („=12). For the fifth group (E4- HFC-R) fed the HF-C diet for 8 weeks and received an intraperitoneal injection of Rinat 5 (3 mg/kg) per week ("12"). After the study was completed, masking for each group: Rinat 3 was 7G10 Rinat 4 is a deglycosylated anti-Aβ antibody 2Η6; and Rinat 5 is 6 G. Fundus examination and luciferin angiography were performed as described previously in Example 4. After the eighth week, as in Example 4 above. The ERG recording was obtained. Results The retinal function was restored/protected by administration of a deglycosylated antibody. As shown in Figure 13, the b-wave amplitude was confirmed to be significant in the group treated with 2H6 (E4-HFC anti-Αβυο). Restoring and/or protecting retinal function. The b-wave amplitude indicates that there is almost no recovery in the group treated with 7G10 (E4-HFC anti-Αβ]-42/Αβ1_43). Surprisingly, the b-wave amplitude is indicated at 6G (E4-HFC anti-ΑβΜο /ΑβΗζ) Treatment 129629.doc • 119- 200900079 The group even more restored and/or protected retinal function. As shown in Figure 14, the group treated with 6G The amplitude of the b-wave can be comparable to that of the control group of normal mice, indicating complete recovery and/or protection of retinal function. Conclusions The above data demonstrate that: 1) the starch of RPE is pathogenic and/or toxic in AMD; 2) Significant recovery/protection of retinal function, as evidenced by ERG in mice injected with anti-Αβ antibody 2H6-D; and 3) complete recovery/protection of retinal function, as injected by bispecific anti-Αβ^ο/Αβ^ι 6G The ERG certification of the mouse. It is to be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art will be able to make various modifications or changes thereto and include them in the present application. In the spirit and scope, all publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes as if the Individual publications, patents, or patent applications are incorporated by reference in their entirety. Biomaterials are preserved by American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 201 10-2209 , USA (ATCC) preserves the following materials: Material Antibody No. ATCC Accession Number Saved Date pDb.9TL.hFc2a 9TL Heavy Chain PTA-6124 July 20, 2010 pEb.9TL.hK 9TL Light Chain PTA-6125 July 20, 2004 Day pDb.6G.hFc2a 6G heavy bond PTA-6786 June 2005 15 pEb.6G.hK 6G light chain PTA-6787 June 2005 15 vector pEb.9TL.hK is a 9TL light chain variable region and a light chain kappa constant region 129629.doc -120-200900079 polynucleotide; and the vector pDb.9TL.hFc2a is a polynucleotide acid encoding a 9TL heavy chain variable region and a heavy bond IgG2a'|·亘定区' The heavy-duty IgG2a region contains the following mutations: A330P331 to S330S331 (refer to the amino acid number of the wild-type lgG2a sequence; see J. (1999) 29:2613-2624). The vector pEb.6G.hK is a polynucleotide encoding a 6G light chain variable region and a light chain busy constant region; and the vector pDb.6G.hFc2a is a polynucleus encoding a 6G heavy bond variable region and a heavy chain IgG2a constant region. Glycosylate, the heavy chain igG2a constant region contains the following mutations: A330P331 to S330S331 (refer to the amino acid number of the wild type lgG2a sequence; see five wr. /. (1999) 29:2613-2624). Such deposits are carried out under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations under the Budapest Treaty. This ensures that the viable culture of the preserves will last for 30 years from the date of storage. The deposit will be paid by the ATCC under the Budapest Treaty and will be subject to an agreement between Rinat Neuroscience Corp. and the ATCC, which will ensure that once the relevant US patent is published or once any US or foreign patent application is filed ( No matter which one appears first) becomes public, the public can obtain permanent and unrestricted use of the children of the culture of the preserve, and ensure compliance with the 35 USC Section 122 and its committee members (including 37 CFR Section) 1.14, its specific reference 886 〇G 638), which is determined by the US Patent and Trademark Commission for its authorization. The assignee of the present application has agreed that if the culture of the preserved material dies or loses or destroys any of the government inventions under the conditions of 129629.doc 121 200900079, then the notice will be immediately notified to another Equal material. The use of the material in question should not be construed as a practice of the law in violation of the rights granted by the authorities under its patent law.

Antibody sequences 9TL heavy chain variable region amino acid sequence (SEQ ID ΝΟ: 1) QVQLVQSGAEVKKPGASVKVSCKASGYYTEAYYIHWVRQAPGQGLE WMGRIDPATGNTKYAPRLQDRVTMTRDTSTSTVYMELSSLRSEDTAV YYCASLYSLPVYWGQGTTVTVSS 9TL light chain variable region amino acid sequence (SEQ ID NO: 2) DWMTQSPLSLPVTLGQPASISCKSSQSLLYSDAKTYLNWFQQRPGQ SPRRLIYQISRLDPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQ GTHYPVLFGQGTRLEIKRT 9TL CDR HI (CDRs of extension) ( SEQ ID NO: 3)

GYYTEAYYIH 9TL CDR H2 (extended CDR) (SEQ ID NO: 4)

RIDPATGNTKYAPRLQD 9TL CDR H3 (extended CDR) (SEQ ID NO: 5)

LYSLPVY 9TL CDR LI (Extended CDR) (SEQ ID NO: 6)

KSSQSLLYSDAKTYLN 9TL CDR L2 (Extended CDR) (SEQ ID NO: 7)

QISRLDP 9TL CDR L3 (Extended CDR) (SEQ ID NO: 8)

LQGTHYPVL

9TL heavy chain variable region nucleotide sequence (SEQ ID NO: 9)

CAGGTGCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGMGCCTGGCGCTTCCGTGA

AGGTTTCCTGCAAAGCATCTGGTTACTATACGGAGGCTTACTATATCCACTGGGTGC

GCCAAGCCCCTGGTCAAGGCCTGGAGTGGATGGGCAGGATTGATCCTGCGACTGG

TAATACTAAATATGCCCCGAGGTTACAGGACCGGGTGACCATGACTCGCGATACCT

CCACCAGCACTGTCTACATGGAACTGAGCTCTCTGCGCTCTGAGGACACTGCTGTG

TATTACTGTGCCTCCCTTTATAGTCTCCCTGTCTACTGGGGCCAGGGTACCACTGTT ACCGTGTCCTCT 9TL light chain variable region nucleotide sequence (SEQ ID NO: 10)

GATGTTGTGATGACCCAGTCCCCACTGTCTTTGCCAGTTACCCTGGGACAACCAG

CCTCCATATCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGCCAAGACATATT

TGAATTGGTTCCAACAGAGGCCTGGCCAGTCTCCACGCCGCCTAATCTATCAGATT

TCCCGGCTGGACCCTGGCGTGCCTGACAGGTTCAGTGGCAGTGGATCAGGCACA -122- 129629.doc 200900079

GATTTTACACTTAAAATCAGCAGAGTGGAGGCTGAAGATGTGGGAGTTTATTACTG

CTTACAAGGTACACATTATCCGGTGCTCTTCGGTCAAGGGACCCGCCTGGAGATC

AAACGCACT 9TL heavy chain full antibody amino acid sequence (including modified IgG2a as described herein) (SEQ ID NO: 11)

QVQLVQSGAEVKKPGASVKVSCKASGYYTEAYYIHVWRQAPGQGLEWMGRIDPATG

NTKYAPRLQDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASLYSLPVYWGQGTTVTV

SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV

AGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWWDGVEVHNAKTKPRE

EQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLP

PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL

TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9TL light chain full antibody amino acid sequence (SEQ ID NO: 12)

DWMTQSPLSLPVTLGQPASISCKSSQSLLYSDAKTYLNWFQQRPGQSPRRLIYQISR

LDPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHYPVLFGQGTRLEIKRTVA

APSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 9TL heavy chain full antibody nucleotide sequence (including modifications as described herein)

IgG2a) (SEQ ID NO: 13)

CAGGTGCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGAAGCCTGGCGCTTCCGTGA

AGGTTTCCTGCAAAGCATCTGGTTACTATACGGAGGCTTACTATATCCACTGGGTG

CGCCAAGCCCCTGGTCAAGGCCTGGAGTGGATGGGCAGGATTGATCCTGCGACTG

GTAATACTAAATATGCCCCGAGGTTACAGGACCGGGTGACCATGACTCGCGATACC

TCCACCAGCACTGTCTACATGGAACTGAGCTCTCTGCGCTCTGAGGACACTGCTGT

GTATTACTGTGCCTCCCTTTATAGTCTCCCTGTCTACTGGGGCCAGGGTACCACTG

TTACCGTGTCCTCTGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGC

TCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACT

TCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCA

CACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGA

CCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA

GCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGT

CCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAA

GCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTG

GACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGG

AGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAG

AGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTAT

AAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAA

GACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGAGAG

GAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATC

CGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACC

ACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGT

GGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAG

GCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAATT

CTAGA 9TL light chain full antibody nucleotide sequence (SEQ ID NO: 14) -123 - 129629.doc 200900079

GATGTTGTGATGACCCAGTCCCCACTGTCTTTGCCAGTTACCCTGGGACAACCAGC

CTCCATATCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGCCAAGACATATTT

GAATTGGTTCCAACAGAGGCCTGGCCAGTCTCCACGCCGCCTAATCTATCAGATTT

CCCGGCTGGACCCTGGCGTGCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAG

ATTTTACACTTAAAATCAGCAGAGTGGAGGCTGAAGATGTGGGAGTTTATTACTGCT

TACAAGGTACACATTATCCGGTGCTCTTCGGTCAAGGGACCCGCCTGGAGATCAAA

CGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAA

ATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCA

AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGT

CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTG

AGCAAAGCAGACTACGAGAAACACAAAG Ding CTACGCC Ding GCGAAGTCACCCATCAGG

GCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAATTCTAG 6G heavy chain variable region amino acid sequence (SEQ ID NO: 26)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYAIHWVRQ

APGQGLEWMGFTSPYSGVSNYNQKFKGRVTMTRDTSTST

VYMELSSLRSEDTAVYYCARFDNYDRGYVRDYWGQGTLV

TVS 6G light chain variable region amino acid sequence (SEQ ID NO: 27) DIVMTQSPDSLAVSLGERATINCRASESVDNDRISFLNW YQQKPGQPPKLLIYAATKQGTGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQSKEFPWSFGGGTKVEIKRTV 6G CDR HI (extended CDR) (SEQ ID NO: 28)

GYTFTTYAIH 6G CDR H2 (Extended CDR) (SEQ ID NO: 29)

FTSPYSGVSNYNQKFKG 6G CDR H3 (extended CDR) (SEQ ID NO: 30)

FDNYDRGYVRDY 6G CDR LI (Extended CDR) (SEQ ID NO: 31)

RASESVDNDRISFLN 6G CDR L2 (Extended CDR) (SEQ ID NO: 32)

AATKQGT 6G CDR L3 (Extended CDR) (SEQ ID NO: 33)

QQSKEFPWS 6G heavy chain variable region nucleotide sequence (SEQ ID NO: 34)

CAGGTGCAACTGGTGCAATCCGGTGCCGAGGTGAAAAAGCCAGGCGCCTCCGTGA

AAGTGTCCTGCAAAGCCTCCGGTTACACCTTTACCACCTATGCCATCCATTGGGTG

CGCCAGGCCCCAGGCCAGGGTCTGGAGTGGATGGGCTTTACTTCCCCCTACTCCG

GGGTGTCGAATTACAATCAGAAGTTCAAAGGCCGCGTCACCATGACCCGCGACACC

TCCACCTCCACAGTGTATATGGAGCTGTCCTCTCTGCGCTCCGAAGACACCGCCGT

GTATTACTGTGCCCGCTTCGACAATTACGATCGCGGCTATGTGCGTGACTATTGGG

GCCAGGGCACCCTGGTCACCGTCTCC 6G light chain variable region nucleotide sequence (SEQ ID NO: 35)

GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCCGTGTCCCTGGGCGAGCGC

GCCACCATCAACTGCCGCGCCAGCGAATCCGTGGATAACGATCGTATTTCCTTTCT

GAACTGGTACCAGCAGAAACCAGGCCAGCCTCCTAAGCTGCTCATTTACGCCGCC

ACCAAACAGGGTACCGGCGTGCCTGACCGCTTCTCCGGCAGCGGTTCCGGCACC

GATTTCACTCTGACCATCTCCTCCCTGCAGGCCGAAGATGTGGCAGTGTATTACTG -124- 129629.doc 200900079

TCAGCAGTCCAAAGAGTTTCCCTGGTCCTTTGGCGGTGGCACCAAGGTGGAGATC

AAACGCACTGTG 6G heavy chain full anti-amidino acid sequence (including modified IgG2a as described herein) (SEQ ID NO: 36)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYAIHWVRQAPGQGLEWMGFTSPYSG VSNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARFDNYDRGYVRDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWWDGVEVHN AKTKPREEQFNSTFRVVSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 6G light chain full antibody amino acid sequence (SEQ ID NO: 37) DIVMTQSPDSLAVSLGERATINCRASESVDNDRISFLNWYQQKPGQPPKLLIYAATKQ

GTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEFPWSFGGGTKVEIKRTVA

APSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 6G heavy chain full antibody nucleotide sequence (including modifications as described herein)

IgG2a) (SEQ ID NO: 38)

CAGGTGCAACTGGTGCAATCCGGTGCCGAGGTGAAAAAGCCAGGCGCCTCCGTGA AAGTGTCCTGCAAAGCCTCCGGTTACACCTTTACCACCTATGCCATCCATTGGGTG CGCCAGGCCCCAGGCCAGGGTCTGGAGTGGATGGGCTTTACTTCCCCCTACTCCG GGGTGTCGAATTACAATCAGAAGTTCAAAGGCCGCGTCACCATGACCCGCGACAC CTCCACCTCCACAGTGTATATGGAGCTGTCCTCTCTGCGCTCCGAAGACACCGCC GTGTATTACTGTGCCCGCTTCGACAATTACGATCGCGGCTATGTGCGTGACTATTG GGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTC TTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGC TCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACT CCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACAC t CTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGA

i AAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCG

TGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAG GTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACT GGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGC AGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTG GCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGC ATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATA CCCTGCCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT GGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAG CCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTT CCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTC TCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTC CCTGTCTCCAGGAAAG 6G light chain full antibody nucleotide sequence (SEQ ID NO: 39)

GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCCGTGTCCCTGGGCGAGCGC

GCCACCATCAACTGCCGCGCCAGCGAATCCGTGGATAACGATCGTATTTCCTTTCT

GAACTGGTACCAGCAGAAACCAGGCCAGCCTCCTAAGCTGCTCATTTACGCCGCC

ACCAAACAGGGTACCGGCGTGCCTGACCGCTTCTCCGGCAGCGGTTCCGGCACC

GATTTCACTCTGACCATCTCCTCCCTGCAGGCCGAAGATGTGGCAGTGTATTACTG -125 - 129629.doc 200900079

TCAGCAGTCCAAAGAGTTTCCCTGGTCCTTTGGCGGTGGCACCAAGGTGGAGATC

AAACGCACTG Ding GGCTGCACCATC Ding GTCTTCATCTTCCCTCCATCTGATGAGCAGU

GAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGG

CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAG

TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACC

CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA

GGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGC m7G10 heavy chain amino acid sequence (SEQ ID NO: 40)

EVKLVESGGDLVKPGGSLKLSCAASGFTFSTYAMSWIRQTPEKRLEWVASIG

NSSRTYYPDSVKGRFTISRDNAGSILYLQMSSLRSEDTAIYYCARGEDGNYAWFT

YWGQGTQVTVS m7G10 light chain amino acid sequence (SEQ ID NO: 41) DIVLTQSPATLSVTPGDSVSLSCRASQSVKNNLHWYQQKSHESPRLLIKYTFQS MSGIPSRFSGSGSGTDFTLIINSVETEDFGMYFCQQSNRWPLTFGAGTKLEL m7G10 HI CDR amino acid sequence (SEQ ID NO: 42)

TYAMS m7G10 H2 CDR amino acid sequence (SEQ ID NO: 43)

SIGNSSRTYYPDSVKG m7G10 H3 CDR amino acid sequence (SEQ ID NO: 44)

GEDGNYAWFTY m7G10 LI amino acid sequence (SEQ ID NO: 45)

RASQSVKNNLH m7G10 L2 Amino Post Sequence (SEQ ID NO: 46)

YTFQSMS m7G10 L3 amino acid sequence (SEQ ID NO: 47)

QQSNRWPLT m7G10HC heavy chain nucleotide sequence (SEQ ID NO: 48)

GAAGTGAAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTG

AAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGCCATGTCTTGGATT

CGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCCTCCATTGGTAATAGTAGTA

GGACTTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCC

GGGAGCATCCTGTACCTCCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATTT

ATTATTGTGCAAGAGGGGAAGATGGTAACTACGCCTGGTTTACTTACTGGGGCCAA

GGGACTCAGGTCACCGTCTCC M7G10HC light chain nucleoside 睃 sequence (SEQ ID NO: 49)

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGT

CAGTCTTTCCTGCAGGGCCAGCCAAAGTGTTAAGAACAACCTACACTGGTATCAAC

AAAAGTCACATGAGTCTCCAAGGCTTCTCATCAAGTATACTTTCCAGTCCATGTCTG

GGATCCCCTCCAGGTTCAGTGGCAGTGGCTCAGGGACAGATTTCACTCTCATTATC

AACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACCGTTG

GCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTG [Simplified Schematic] Figure 1 shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 1) and the light chain variable region (SEQ ID NO: 2) of the 9TL antibody. The Kabat CDR lines are in bold and the Chothia CDR lines are underlined. Amines for the heavy and light chain variable regions -126- 129629.doc 200900079 The acid residues are numbered sequentially. Figure 2 shows the epitope localization of antibody 9TL by peptide competition. The peptide was immobilized on a SA wafer. And then each with 10 μM of various peptides (amino acids 28-40, 1-40, 1-28, 28-42, 22-35, 1-16, ΙΑ, 33-40, 1-38 or 17-40) or a single antibody 2289 and a 9TL Fab fragment (each 50 nM) pre-incubated for 1 h without peptide flow on the wafer. The binding of the antibody Fab fragment to the immobilized ΑβΝ40 peptide was measured. Figure 3 is a graph showing the epitope localization of antibody 2Η6 by peptide competition. The Αβ^o peptide was immobilized on a SA wafer. Each of 16 μΜ of various peptides (amino acids 1-16, 1-28, 1-38, 1-40, 1-42, 1-43, 17-40, 17-42, 22-3 5, 25-35 or 33-40) or single antibody 2289, 228 6 or 2H6 (100 nM each) pre-incubated for 1 h without peptide flow on the wafer. The binding of the antibody to the immobilized peptide is measured. Figure 4 is a graph showing the binding of antibodies 2Η6, 2286 and 2289 to different Αβ peptide C-terminal variants. GST-Αβ variants (Μ35Α, V36A, G37A, G38A, V39A or V40A) or GST-Αβ peptides 1-3 9 , 1-41, 1-40, 1-42 were immobilized on an ELISA plate. Monoclonal antibodies 2286, 2H6 or 2289 (0.3 mM each) were incubated with each of the immobilized peptides, and their binding was detected by further biotinylated anti-mouse IgG (H+L) and Sterptavidin-HRP. Figure 5 is a graph showing the intensity of a-wave and b-wave (A) and electroretinogram (B) from a normal lipoprotein isoform E4 (APOE4) mouse in a normal diet versus a high fat and cholesterol diet. Figure 6 is a graph showing the intensity of the b-wave of the mouse only in the previous study of the normal diet animals compared to the APOE4 small 129629.doc -127- 200900079. The R2 trace shows protection or recovery of retinal function when AMD-like mice (E4-HFC-R2) are treated with anti-Aβ antibodies. Figure 7 shows the total Αβ immunohistochemistry of the brain of AMD-like (ΑΡΟΕ4) mice. Slide Α (AMD-like mice treated with anti-Aβ antibody) showed negative starch detection. Slides B, C, and D (AMD-like mice treated with vehicle injection) exhibited positive starch-like detection. Slide E was taken from a positive control and was obtained from a platelet-derived APP mouse model (pdAPP, a mutation (V717F) human APP under the control of a platelet-derived growth factor promoter (Games, D. et al., Nature 373: 523- Brain of 527 (1995)) Figure 8 shows the amino acid sequence of the heavy chain variable region (SEQ ID ΝΟ: 1) and the light chain variable region (SEQ ID NO: 27) of the 6G antibody. The Kabat CDR system is coarse The Chothia CDRs are underlined and the amino acid residues of the heavy and light chain variable regions are numbered sequentially. Figure 9 shows the epitope localization of antibody 6G by ELISA. 16, 1-28, 17-40, 17-42, 22-35, 28-40, 28-42, 1-38, 1-40, 1-42, 1-43 and 33-40) fixed to the ELISA plate Monoclonal antibody 6G (20 nM) was incubated for 1 h with various immobilized peptides. Antibody anti-human kHRP-conjugated secondary antibody was used to measure antibody 6G bound to immobilized Aβ peptide. Figure 10 shows antibody 6G by ELISA Site determinant immobilization of various Αβ peptides on ELIS A plates. Antibody 6G was incubated for 1 h with various immobilized peptides. Measurement and immobilization using goat anti-human kHRP conjugated secondary antibodies The β-peptide-conjugated antibody 6G.ΠΝΒ” means that no binding was detected. Figure 11 is a schematic diagram showing the epitope of antibody 6G binding on Aβ. 129629.doc -128- 200900079 Demonstrates the Αβ-like powder precursor protein (APP) The relative position of the part of the APP in the cell membrane. &quot;CT99&quot; refers to the 99 amino acid at the C-terminus of APP. Figure 12 is a photograph showing the immunostaining of APP-expressing cells by monoclonal antibody against antibody 6G. The cells in the row display cells were cultured at m2324 or 6G (5 pg/ml each) and the cells under the fluorescence microscope were detected by binding of goat anti-mouse or anti-human antibodies by secondary Cy3. The bottom row was displayed under a microscope. The cells observed.

Figure 13 is a b-wave intensity map of APOE4 mice in only five study groups: normal dietary control APOE4 mice; high fat and cholesterol diet ('HFC,) control APOE4 mice (AMD-like model); treated with 7G10 APOE4-HFC mice; APOE4-HFC mice treated with 2H6; and APOE4-HFC mice treated with 6G. Figure 14 is a b-wave intensity map of APOE4 mice in only three study groups: normal dietary control APOE4 mice; control APOE4 HFC mice; and 6 g treated APOE4-HFC mice. 129629.doc -129-

Claims (1)

200900079, the scope of application for patents: 1. The use of a class of β; the inhibitor of the powder of the temple (Αβ) peptide 瘩·古Β ... uses ' is used to manufacture drugs for the treatment of individuals with heart disease. 2. 3. The use of the inhibitor, wherein the inhibitor is an antibody that specifically binds to a Αβ peptide selected from the group consisting of: in”, 丨-38, Αβ, _39, Αβ, -40, Αβ丨·42 and from 43. (4), wherein the antibody specifically binds to an epitope of U. 4. The use of claim 3, wherein the antibody is also determined by the antigen of u The specificity of the combination. 5. The use of claim 1, wherein the disease is age-related macular degeneration. 6. The use of claim 2, wherein the antibody is about 1 〇〇 or less and the Αβ 7. The peptide according to claim 3, wherein the antibody binds to the peptide with a Kd of about 1 〇〇 Μ or below. 8. The use of claim 8, wherein the antibody is also about ι 〇〇 or The following Kd binds to the Αβ丨·42 peptide. 9. The use of the item 2, wherein the antibody binds to the 匚β peptide. The use of the epoch 2, wherein the antibody comprises an amino acid 25_34 and 40 epitope binding. 11. The use of the item 2, wherein the antibody is higher than it and AP1 42 and Αβι, ° σ Affinity binds to Αβΐ-40, and wherein the antibody is not antibody 2294 〇12. The use of claim 2, wherein the Fc region of the antibody is not Ν-glycosylated or 129629.doc 200900079 has relative to the native Fc region The altered N-glycosylation pattern. 13. The use of claim 2, wherein the antibody comprises a heavy chain variable region comprising the antibody 6G heavy chain variable region set forth in SEQ ID N:26 Three CDRs, and a light chain variable region comprising three CDRs of the antibody 6G light chain variable region set forth in SEQ m N〇: 27. 14. The use of claim 2, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26, J and a light chain variable region comprising the aminopurine sequence shown in SEQ ID NO: 27. The use of an epitope-specific X-spot degradation on Αβι.4〇 and Αβ〗 _42 for the manufacture of a medicament for treating an individual having age-related m. 129629.doc